Dicty News Electronic Edition Volume 10, number 16 June 27, 1998 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 =========== A New Way of Following Individual Cells in the Migrating Slugs of Dictyostelium discoideum J. T. Bonner Department of Ecology and Evolutionary Biology, Princeton University, Princeton. NJ 08544 PNAS, in press. Abstract: In the development of the cellular slime mold D. discoideum there is a stage in which the aggregated amoebae form a migrating slug that moves forward in a polar fashion, showing sensitive orientation to environmental cues, as well as early signs of differentiation into anterior prestalk and posterior prespore cells. Heretofore it has been difficult to follow the movement of the individual cells within the slug, but a new method is described in which small, flat (one cell thick) slugs are produced in a glass-mineral oil interface where one can follow the movement of all the cells. Observations of time lapse videos reveal the following facts about slug migration: 1) While the posterior cells move straight forward, the anterior cells swirl about rapidly in a chaotic fashion. 2) Turning involves shifting the high point of these hyperactive cells. 3) Both the anterior and the posterior cells move forward on their own power as the slug moves forward. 4) There are no visible regular oscillations within the slug. 5) The number of prestalk and prespore cells is proportional for a range of sizes of these mini slugs. All of these observations on thin slugs are consistent with what one finds in normal, three dimensional slugs. ------------------------------------------------------------------------- Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivaton of genes in Dictyostelium F. Płta, CJ. Zeng* Department of Physiology and Developmental Biology, Charles University, Praha, Czech Republic; Department of Genetics, University of Kassel, Kassel, Germany Folia Biologica, in press ABSTRACT In Dictyostelium discoideum inactivation of developmentally regulated genes via homologous recombination has become an important tool in studying systematically the entire developmental program of this model organism. The Dictyostelium genome is very A/T-rich, however, which presents obstacles to the preparation of knockout constructs. The coding regions offer few suitable restriction sites and the low complexity intergenic regions do not guarantee specificity of recombination. We present here the preparation of plasmids pBsR479, pBsR503, and pBsR519, in which a blasticidin resistance-cassette is positioned in the center of various symmetrical polylinkers. This design simplifies the cloning process and gives more flexibility in positioning the selectable marker within the coding regions. ------------------------------------------------------------------------- Detection of subtles phenotypes: the case of the cell adhesion molecule csA in Dictyostelium. Eleonora Ponte*, Enrico Bracco*, Jan Faix# and Salvatore Bozzaro*Ż *Dipartimento di Scienze Cliniche e Biologiche, Universitą di Torino - Ospedale S. Luigi, 10043-Orbassano (Italy) and #Max-Planck-Institut fuer Biochemie, D-82152 Martinsried (Federal Republic of Germany PNAS, in press ABSTRACT Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally-regulated and triggered by homophilic interactions of the membrane glycoprotein csA; upon disruption of the encoding gene, EDTA- resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development, and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum and slower motility, which lead to their sorting out from aggregating wild- type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The new assay described can be used to detect subtle phenotypes, to re-examine the developmental role of apparently non-essential genes and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy. ------------------------------------------------------------------------- Overexpression of CAF1 encoding a novel Ca2+-binding protein stimulates the transition of Dictyostelium cells from growth to differentiation Mikiko Itoh, Maiko Noguchi and Yasuo Maeda* Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan. Develop. Growth & Differ., in press SUMMARY Among genes expressed associated with the switch-over of Dictyostelium cells from cell proliferation to differentiation, the Calfumirin-1 (CAF1) gene has been shown to be preferentially expressed at the initial step of differentiation and encode a novel Ca2+-binding protein (Abe & Maeda 1995). To analyze precisely the function of CAF1, transformants overexpressing the CAF1 mRNA a t the vegetative growth phase and also CAF1-null mutants were prepared, and their developmental features were compared with those of parental wild-type cells. As a result, the CAF1-overexpression was found to promote cell differ entiation, possibly through prompt induction of the cAMP receptor 1 (CAR1) g ene expression. In addition, the CAF1- overexpressing cells were able to diff erentiate even under low external Ca2+ ([Ca2+]e) conditions around 10-6M at which nontransformed wild-type cells never differentiated. Unexpectedly, how ever, the CAF1-null mutant produced by homologous recombination exhibited ap parently normal development to form fruiting bodies on non-nutrient agar. These results seems to indicate that the CAF1-overexpression has a stimulatory effect on differentiation, but that the CAF1 protein is not necessarily required for the phase-shift of cells from growth to differentiation. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 16]