CSM News Electronic Edition Volume 7, number 5 August 31, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =============== Announcements =============== Bill Loomis writes: I made a table of our cDNA sequences that we have deposited in GenBank during the last month. They are derived from probes used in the Mapping Project and so provide sequence tagged sites for whole genome sequencing. Moreover, the Dicty community might be interested to see what is out there now. Perhaps you could put this on the next Dicty e-mail. Some of these genes are old friends while others are new and are very interesting. A few are the subjects of on-going genetic studies. Others are not so informative but it is good to have sequence for them. Bill cDNAs Sequences of Mapped Loci Previous New Locus Sequence homology Map position locus Genbank No. rcdA WD40 prot 2 3.162 wdpA U36936 rcdB phosphoprotein phosphatase 2A 4.109 pppA U61987 rcdC coatomer beta subunit 4.142 copB U62960 rcdD cadA (gp24) 4.177 cadA U49650 rcdE phosphoglucomutase 5.028 pgmA U61984 rcdF ribosomal protein S17 5.118 rpgM P42520 rcdG ubiquinone oxido-reductase 5.046 qinA U61989 rcdH carbonic anhydrase 5.086 canA U66368 rcdJ cigA (BP74) 2.012 cigB U66528 rcdK none 2.032 --- U66369 rcdL none 2.035 --- U66366 rcdM none 2.058 --- U66527 rcdP none 1.316 --- U66525 rcdQ PEP 8 rel. (protein sorting) 1.336 pepA U61985 rcdR cyclophilin 1.358 ppiA 227879 rcdS proliferation assoc. protein 1.360 prlA U61403 rcdX proteasome C8 1.005 prtD U61988 rcdBB none 1.316 --- U66523 rcdEE hsp70 1.336 hspB L22736 rcdFF protein kinase C inhibitor 5.157 pkiA U61986 rcdHH saposin B[low similarity] 4.053 sapA U66367 rcdII none 4.051 --- U66524 rcdJJ none 4.155 --- U66370 rcdKK thymidine kinase 5.067 thyB U61990 rcdLL ubiquitin 2.233 ubqG 70660 =========== Abstracts =========== Vacuolar H+-ATPase and weak base action in Dictyostelium. L. Davies 1, N.A. Farrar 1, M. Satre 2, R.P. Dottin 3 and J.D. Gross 1# 1 Department of Biochemistry, South Parks Road, Oxford, UK. OX1 3QU. 2 Laboratoire de Biologie Cellulaire (CNRS URA 1130), Departement de Biologie Moléculaire et Structurale, CEA, 38041 Grenoble, France 3Department of Biological Sciences, Hunter College, City University of New York, 695 Park Avenue, New York 10021, USA. Mol. Microbiology, in press Summary Amoebae of Dictyostelium discoideum release ammonia during development, and the accumulation of this weak base is believed to be responsible for inhibiting fruiting body formation and switching aggregates into migrating slugs. Exposure to weak bases can also inhibit aggregation and cell-type specific gene expression. The pathway by which weak bases influence development is not understood. We show here that the development of a set of mutants defective in acidification of intracellular acidic compartments is abnormally sensitive to inhibition by weak bases. Moreover even in the absence of added weak bases these mutants are delayed in aggregation and have a protracted migratory phase. The same behaviour is observed in transformants harbouring an antisense construct for one of the vacuolar H+-ATPase subunits.These results support the idea that weak bases exert their effects by inhibiting acidification of an intracellular acidic compartment. --------------------------------------------------------------------- A role for calcineurin in Dictyostelium discoideum development. Fabiana Horn and Julian Gross Department of Biochemistry,University of Oxford.South Parks Road Oxford OX1 3QU, UK. Differentiation, in press Abstract We have used the immunosuppressants cyclosporin A and FK506 to investigate the involvement of the Ca2+/ CaM-dependent protein phosphatase calcineurin in D. discoideum development. We found that CsA had little effect on cell growth, or on the aggregation of developing amoebae, suggesting that calcineurin does not play a significant role at these stages of the D. discoideum life cycle. In contrast, when cells were allowed to differentiate under buffer in the presence of cAMP, addition of CsA and FK506 strongly inhibited stalk cell formation in the wild type and spore formation in a sporogenous derivative of D. discoideum strain V12. These agents also reduced the expression of prestalk- and prespore-specific transcripts in both strains. These results indicate a requirement for calcineurin activity in both pathways of cell differentiation. In addition, time-course experiments suggest that calcineurin is required early in the differentiation processes, but that the maturation of the two cell types is resistant to calcineurin inhibition. We also found that CsA and FK506 were unable to affect spore formation in rapidly developing/sporogenous rdeC mutants of strain NC4, showing that constitutive cAMP-dependent protein kinase activity renders the spore pathway resistant to calcineurin inhibition. --------------------------------------------------------------------- Cloning and Characterization of a Dictyostelium Myosin I Heavy Chain Kinase Activated by Cdc42 and Rac Sheu-Fen Lee, Thomas T. Egelhoff*, Amjad Mahasneh and Graham P.Cote Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6 and *Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 J. Biol. Chem. In Press SUMMARY The motile activities of the small, single-headed class I myosins (myosin I) from the lower eukaryotes Acanthamoeba and Dictyostelium are activated by phosphorylation of a single serine or threonine residue in the head domain of the heavy chain. Recently, we purified a myosin I heavy chain kinase (MIHCK) from Dictyostelium based on its ability to activate the Dictyostelium myosin ID isozyme (Lee, S.-F. and Cote, G.P. (1995) J. Biol. Chem. 270, 11776-11782). The complete sequence of the Dictyostelium MIHCK has now been determined, revealing a protein of 98 kDa that is composed of an amino terminal domain rich in proline, glutamine and serine, a putative Cdc42/Rac binding motif and a carboxyl-terminal kinase catalytic domain. MIHCK shares significant sequence identity with the S. cerevisiae Ste20p kinase and the mammalian p21-activated kinase (PAK). Gel overlay assays and affinity chromatography experiments showed that MIHCK interacted with GTPgammaS (guanosine5'-3-O-(thiotriphosphate))- labeled Cdc42 and Rac1 but not RhoA. In the presence of GTPgammaS-Rac1 MIHCK autophosphorylation increased from 1 to 9 mol phosphate per mol and the rate of Dictyostelium myosin ID phosphorylation was stimulated 10-fold. MIHCK may therefore provide a direct link between Cdc42/Rac signaling pathways and motile processes driven by myosin I molecules. --------------------------------------------------------------------- A nuclear protein factor binds specifically to the 3+ regulatory module of the long-interspersed-nuclear-element-like Dictyostelium repetitive element Anja Geier, Jens Horn, Theodor Dingermann and Thomas Winckler Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany Eur. J. Biochem., in press SUMMARY The Dictyostelium repetitive element DRE integrates in a position-specific manner upstream of tRNA genes in the Dictyostelium discoideum genome. DRE has structural similarities to the group of long interspersed nuclear elements, whose replication mechanism is poorly understood. The C-module at the 3' end of DRE encodes a regulatory cis-acting sequence that contains an RNA polymerase II promoter. This promoter directs the synthesis of RNAs that are thought to play a critical role in DRE transposition. In this study, we describe the identification of a nuclear protein factor that binds to the C-module in a sequence-specific manner. The C-module-binding factor (CMBF) recognizes three DNA sequence motifs that contain homopolymeric (dT) stretches of variable lengths, but does not bind to a standard RNA polymerase II promoter from D. discoideum. Analysis of highly CMBF-enriched fractions and glycerol gradient sedimentation of CMBF suggest that the factor exists as a monomeric 115 kDa protein. Possible roles of CMBF in DRE transposition are discussed. --------------------------------------------------------------------- [End CSM-News, volume 7, number 5]