CSM News Electronic Edition Volume 5, number 3 July 22, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" ==================== Position Available ==================== POST-DOCTORAL POSITIONS REGULATION OF SIGNAL TRANSDUCTION AND PATTERN FORMATION Several post-doctoral fellowships are available to study the role of the cAMP receptors and downstream pathways in control of Dictyostelium development and regulated gene expression. Experience in molecular biology, protein chemistry or receptor biochemistry is essential. Applicants must have less than five years of post-doctoral experience; US citizenship is not required. Please send curriculum vitae and the names, addresses and telephone numbers of three references to: Dr. Alan R. Kimmel Building 6; Room B1-22 LCDB/NIDDK NATIONAL INSTITUTES OF HEALTH Bethesda, MD 20892-2715 USA Tel.: 301-496-3016; Fax: 301-496-5239; e-mail:ark1@helix.nih.gov =========== Abstracts =========== Transformation with vectors harbouring the NEOR selection marker induces germination specific adenylylcyclase activity in Dictyostelium cells. Conchita Schulkes, Irene Verkerke-van Wijk and Pauline Schaap. Cell Biology Section, Institute of Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Exper. Cell Res., in press. SUMMARY Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the aminoglycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells. --------------------------------------------------------------------- Dictyostelium discoideum mitochondrial DNA enclodes a NADH:ubiquinone oxidoreductase subunit which is nuclear encoded in other eukaryotes Robert A. Cole, Martin B. Slade and Keith L. Williams (1995) Journal of Molecular Evolution 40:616-621 Summary Complex I, a key component of the mitochondrial electron transport system, is thought to have evolved from at least two separate enzyme systems prior to the evolution of mitochondria from a bacterial endosymbiont, but the genes for one of the enzyme systems are thought to have subsequently been transferred to the nuclear DNA. We demonstrated that the cellular slime mould Dictyostelium discoideum retains the ancestral characteristic of having mitochondria encoding at least one gene (80kDa subunit) that is nuclear encoded in other eukaryotes. This is consistent with the cellular slime moulds of the family Dictyosteliacae having diverged from other eukaryotes at an early stage prior to the loss of the mitochondrial gene in the lineage giving rise to plants and animals. The D. discoideum mitochondrially encoded 80 kDa subunit of complex I exhibits a two fold higher mutation rate compared with the homologous chromosomal gene in other eukaryotes, making it the most divergent eukaryotic from of this protein. ----------------------------------------------------------------------- Expression of CSA-hm2 fusion in Dictyostelium discoideum under the control of the Dictyostelium ras promoter reveals functional muscarinic receptors. Guido Voith and Theodor Dingermann Institut for Parmaceutical Biology, Johann Wolfgang Goethe University, Biozentrum, Marie-Curie-Str. 9, D-60439 Frankfurt/Main, Germany PHARMAZIE, in press Summary We have expressed the human m2 muscarinic receptor gene in the cellular slime mold Dictyostelium discoideum. Expression under the control of the constitutive actin 6 promoter without a D. discoideum leader peptide results in cells which seem to respond to muscarinic agonists initially, but which quickly revert to non responding cells only after a few generations. However, when expressing the hm2 gene as a fusion gene together with the CSA leader peptide under the control of the regulated D. discoideum ras promoter cells are obtained which express functional muscarinic M2 receptors in a stable manner. As expected from the typical regulation of the ras promoter, M2 receptors are expressed only during development. In ligand binding assays these heterologously expressed receptors show binding characteristics similar to authentic M2 receptors. ----------------------------------------------------------------------- Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries. J. T. Bonner*, Katharine B. Compton@, Edward C. Cox@, Petra Fey@ and Keqin Y. Gregg@ Department of Ecology and Evolutionary Biology* and the Department of Molecular Biology@, Princeton University, Princeton, NJ 08544 Proc. Natl. Acad. Sci. USA, in press ABSTRACT When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps towards the formation of prestalk and prespore zones. Some of the events occur within a minute or two, while others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different sized cell masses, and those proportions are the same as those found in normal slugs. If the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein regulated by a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells. ---------------------------------------------------------------------- Green fluorescent protein production in the cellular slime molds Polysphondylium pallidum and Dictyostelium discoideum Petra Fey, Kate Compton and Edward C. Cox Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA. Tel. (609) 258-3571 GENE, in press SUMMARY The green fluorescent protein-encoding gene from Aequorea victoria has been cloned into several different transforming vectors and expressed in the cellular slime molds, Polysphondylium pallidum and Dictyostelium discoideum. We find that the protein is stable and non-toxic in both species, can be easily visualized in living and fixed specimens, and can be used to purify rare cells by fluorescence activated cell sorting. ----------------------------------------------------------------------- [End CSM News, volume 5, number 3]