CSM News Electronic Edition Volume 3, number 17 November 26, 1994 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 through www.nwu.edu. ------------------------------------------------------------------ Glycogen synthase kinase 3 (GSK-3) regulates cell fate in Dictyostelium A.J. Harwood, S. E. Plyte*, J. Woodgett*, H. Strutt+, & R.R.Kay MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, CB2 2QH, U.K. *Ontario Cancer Institute, Princes Margaret Hospital, 500 Sherbourne St. Toronto, Ontario, Canada, M4X 1K9 Cell, in press. Abstract Extracellular cAMP induces the formation of prespore cells in Dictyostelium but inhibits stalk cell formation. We have cloned, gskA, the Dictyostelium homologue of the protein kinase GSK-3 and discovered that it is required for both cAMP effects. Disruption of gskA creates a mutant that aggregates but forms few spores and an abnormally high number of stalk cells. These stalk cells probably arise from an expanded prestalk B (pstB) cell population, which normally produces the basal disc of the fruiting body. In cultured mutant cells cAMP neither inhibits pstB cell differentiation nor induces efficient prespore cell differentiation. We propose that cAMP acts through a common pathway that requires GSK-3 and determines the proportion of prespore and pstB cells. ------------------------------------------------------------------------ The proximal pathway of metabolism of the chlorinated signal molecule DIF-1 in Dictyostelium Piero Morandini, John Offer, David Traynor, Oliver Nayler, David Neuhaus, Graham W. Taylor* and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK *Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W12 OHS. Present addresses. PM: Dipartimento di Biologia 'L. Gorini", via Celoria 26, 20133 Milano, Italy. DT: Department of Pharmacology, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QJ, UK Biochemical J., in press. Abstract Stalk cell differentiation during Dictyostelium development is induced by a chlorinated alkyl phenone called DIF-1. Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to a dozen unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by mass spectroscopy, NMR and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DM1. DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolised by three successive oxidations of its aliphatic side chain: a hydroxylation at w-2 to produce DM2, oxidation of the hydroxyl to a ketone to produce DM3 and a further hydroxylation at w-1 to produce DM4, a hydroxy ketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side chain oxidation was detected by incubating 3H-DIF-3 with cell-free extracts and resolving the reaction products by TLC. It will be referred to as DIF-3 hydroxylase. DIF-3 hydroxylase has many of the properties of a cytochrome P450. It is membrane bound and uses NADPH as co-substrate. It is also inhibited by carbon monoxide, the classic P450 inhibitor, and by several other P450 inhibitors as well as by diphenyliodonium chloride, an inhibitor of cytochrome P450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, except for a DIF-3 analog lacking the chlorine atom. The Km for DIF-3 of 47nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50=67nM) will be particularly suitable for probing the function of DIF metabolism during development. ------------------------------------------------------------------ ISOLATION OF DICTYOSTELIUM DISCOIDEUM CYTOKINESIS MUTANTS BY RESTRICTION ENZYME-MEDIATED INTEGRATION OF THE BLASTICIDIN S RESISTANCE MARKER Hiroyuki Adachi1,*, Takeshi Hasebe2, Keisuke Yoshinaga1, Takahisa Ohta2 and Kazuo Sutoh1 1Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153, and 2Department of Applied Chemistry, Faculty of Engineering, Kogakuin University, Shinjuku-ku, Tokyo 163-91, Japan *Corresponding author; aduc@tansei.cc.u-tokyo.ac.jp BBRC, in press. Summary We have developed an improved REMI (restriction enzyme-mediated integration) system for generating mutant Dictyostelium cells quickly and efficiently for systematic screening of cytokinesis mutants. By means of this system, three cytokinesis mutants that grow as giant and multinucleate cells were isolated from 2,000 Dictyostelium transformants. Southern blot analysis of these mutants revealed that a single copy of the tag DNA was integrated into each genome. The tag with flanking genomic DNA at both ends was rescued from one of the mutants, and reintroduced into the parental Ax2 strain. Homologous recombination of the rescued gene and the Dictyostelium genome led to the phenotypical changes expected for cytokinesis mutants. ------------------------------------------------------------------- [End CSM News, volume 3, number 17]