CSM News Electronic Edition Volume 3, number 19 December 17, 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. --------------------- Positions Available --------------------- C1 position in Genetics/Molecular Biology available (postdoc, max. 5 years, possibility for habilitation) at the Dept. of Genetics, University of Kassel, Germany, This is a new laboratory which is currently being set up and should be running by April/May 1995. Nevertheless, the successful applicant will be required to participate in the initial establishment and organization. The lab will be well equipped and sufficiently funded. Applicants should be highly motivated and capable to establish a semi- independent research project within the frame outlined below. Application for extramural funding is strongly encouraged and could provide resources for setting up a small group. C1 positions require some participation in teaching, therefore knowledge in German language and/or commitment to learn German would be an advantage. The university is an equal opportunity employer and strongly encourages the application of women. Research projects: 1. Signal transduction/gene regulation in Dictyostelium We are using REMI (restriction enzyme mediated integration) to identify components of the signalling chains leading to regulation of early developmental genes. 2. Mechanisms of antisense mediated gene silencing in Dictyostelium We are trying to identify potential components of the endogenous antisense machinery (dsRNase, RNA binding proteins, etc.) and test their function in antisense mechanisms. 3. Antisense mechanisms in plants In a new project we will extend the work on antisense mechanisms to the plant field (probably tobacco and/or Arabidopsis) using our knowledge (and material) from the Dictyostelium project as a basis. References of recent papers can be provided upon request. Interested individuals should contact me now by e-mail (address below) providing a CV, names of two references and a brief summary of research experience and the current work. I will pre-select candidates and advice them when and where to send the official application. This may seem complicated but is actually a shortcut in German burocracy! wolfgang nellen MPI Biochem. D-82152 Martinsried Germany Tel. ++49 (89) 8578 2241 FAX ++49 (89) 8578 3777 nellen@vms.biochem.mpg.de ------------------------------------------------------------------------- I have an immediate opening for a postdoctoral associate here at the La Jolla Cancer Research Foundation. There are several projects that involve molecular aspects of Glycobiology in Dictyostelium. These include mostly investigation of phosphoglycosylation, a novel form of glycosylation in Dicty. We are trying to identify glycoyl transferase genes, and to biochemically characterize these transferases as well.A background in molecular aspects of Dicty is essential and some knowledge of Glycobiology would be helpful. A competitive salary and benefit package is available. Interested candidates should contact me by e-mail, FAX or letter including a CV and the names of at least three references. Send by mail to : Hud Freeze, Senior Staff Scientist La Jolla Cancer Research Foundation 10901 N. Torrey Pines Rd. La Jolla CA 92037 PHONE: 619-455-6480 ----------- Abstracts ----------- A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain B.W.Wetterauer1, U. Hamker, A. von Haeseler, H.K. MacWilliams, M.-N. Simon and M. Veron BBA, in press We report the sequence of a protein kinase (DdKinX) from Dictyostelium discoideum. It codes for a protein of 1093 amino acids, which are organized in four regions (A) the N-terminal catalytic domain, (B) a region containing 30% acidic amino acids, (C) a region which consists of tandem repeats of the motif PVKVEEPVEE or variants of it and (D) the C-terminus which is again rich in acidic amino acids and serine, threonine and proline residues. The N-terminal sequence is in good agreement with the consensus motifs for myristylation and the C-terminus might be prenylated. DdKinX is weakly expressed. Similarity to other protein kinases is low except for the short consensus motifs (25 to 30% identity). Descendent trees calculated with the catalytic core show that the branch leading to DdKinX evolved independently from other kinase families. The root of the branch lies between mos and raf serine-threonine kinases, tyrosine kinases and the other serine-threonine kinases. --------------------------------------------------------------- Multiple Signal Transduction Pathways Regulate Discoidin I Gene Expression in Dictyostelium discoideum Jurgen Blusch1, Stephen Alexander2 and Wolfgang Nellen1 1 Max-Planck-Institut fur Biochemie, Abteilung Zellbiologie, D-82152, Martinsried, Germany 2 Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211 USA Differentiation, in press Abstract. The expression of the discoidin I genes in Dictyostelium discoideum is regulated by the concerted action of the extracellular factors cAMP, folate, PSF (Prestarvation Factor) and CMF (Conditioned Media Factor). However, the pathways by which these signals are transduced and the interactions between the pathways were unexplored. We have analysed wild-type and mutant cells with defined lesions in signal transduction to elucidate these regulatory processes. We show that different pathways are used for the down-regulation and induction of these genes. The cAMP receptor cARI is required for the cAMP mediated down-regulation of discoidin I gene expression but not for the induction of the expression during development. Surprisingly, the induction of the discoidin I genes requires Ga2, the G-protein subunit which is generally believed to couple to cARI, to control the expression of cAMP inducible genes. Thus, our data suggests that Ga2 interacts with different receptors to regulate gene expression in early development. Furthermore, the analysis shows that discoidin induction in bacterially grown cells occurs in two sequential steps. The first is a low basal induction which occurs in late-log phase growth prior to starvation. PSF can induce the basal level and the induction is independent of Ga2. The developmental induction following starvation is much stronger, dependent on Ga2 and probably signaled by CMF which is secreted at this time. We also demonstrate that the growth history of the cells has a major influence on the pattern of gene expression and on the choice of the signal transduction pathway that is used to induce the discoidin I genes. Overall, these studies reveal complex regulatory interactions between the stimulatory and inhibitory signaling pathways controlling the expression of the discoidin I gene family. ------------------------------------------------------------------- Growth and developmental functions of a human HIV-Tat binding protein/26S protease subunit homologue from Dictyostelium discoideum Jie-Gang Cao and Richard A Firtel Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634. telephone: 619-534-2788, fax: 619-534-7073 Mol. Cell. Biol., in press. ABSTRACT We have characterized a newly identified gene from Dictyostelium, DdTBPa, that encodes a member of the family of eukaryotic proteins containing a conserved ATPase domain that include subunits of the 26S protease subunit and are homologous to the mammalian HIV-Tat binding protein TBP1. While information indicates that some family members are involved in regulating transcription during growth in mammalian cells and yeast, these proteins are also involved in other cellular functions, and nothing is known about their possible function in multicellular development. The Dictyostelium DdTBPa gene is developmentally regulated, being expressed at the highest levels during growth and early development. The gene is present in two copies in the genome. Disruption of one copy by homologous recombination leads to aberrant morphogenesis from the formation of the first finger until the onset of culmination. The gene appears to be essential for growth since we were unable to obtain a complete null phenotype and expression of an inducible antisense construct in the partial null resulted in cell death. Expression of the antisense construct during development accentuated the partial null phenotype and also resulted in very abnormal fruiting bodies. Overexpression of DdTBPa from its own promoter leads to very large multinucleated vegetative cells when the cells are grown in suspension culture. When plated onto Petri dishes in growth medium, the cells rapidly split into multiple cells containing 1-2 nuclei, similar to wild-type cells. Overexpressing cells are significantly delayed in forming a multicellular aggregate, but development proceeds normally once the first finger stage is reached. The results indicate that DdTBPa plays an important role in regulating both growth and morphogenesis in Dictyostelium. ----------------------------------------------------------------------- Glycoprotein complexes interacting with cellulose in the "cell print" zones of the Dictyostelium discoideum extracellular matrix. Ti Zhou-Chou, Wilkins, M.R., Vardy, P.H., Gooley, A.A. & Williams, K.L. Developmental Biology, in press Abstract Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which co-localize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa; where the monomers are 12-35 kDa. The sheathin subunits are similar but not identifical proteins. The sheathin family comprises sheathin 68 (68 kDa trimer); sheathin 62, kDa dimer); sheathin 55,(55kDa dimer) and sheathin 53,(53kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD and ShE which are linked by disulphide bonds. Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino-terminal sequences. This group of D.discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D.discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, eg. SDS or 8 M urea. Sheathins from a compnent of the "cell prints" which are believed to have a role in cell- ECM interations and slug cell migration. ----------------------------------------------------------------------- Immunochemical, Genetic and Morphological Comparison of Fucosylation Mutants of Dictyostelium discoideum Champion, A., Griffiths, K., Gooley, A.A., Gonzalez, B.Y., Gritzali, M., West, C.M. & Williams, K.L. Abstract Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described; mutations in two of these loci result in the simultaneous loss of two separate carbohydrate epitopes. The Ga-X epitope, which is competed by L-fucose, is absent in strains carrying a modC354, modD352 or modE353 mutation. These strains expose a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins is reduced. A second epitope (GA-XII) is also absent in strains carrying the modC354 or mod E353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content is reduced in the three mutants, suggesting that each mutation affects a separate step in fucosylation. The lesions do not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles are more active than normal vesicles at transferring (14C) fucose from GDP-(14C) fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporate exogenous (3H) fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intact modE353 mutant vesicles are relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens CGA-X and GA-XII in this mutant). Finally, the modC354 mutation leads to delayed accumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D.discoideum consisting of defined biochemical steps which contribute to multicellular morphogenesis in this organism. ---------------------------------------------------------------------- The pH-sensitive actin-binding protein hisactophilin of Dictyostelium exists in two isoforms which both are myristylated and distributed between plasma membrane and cytoplasm Frank Hanakam, Christoph Eckerskorn, Friedrich Lottspeich, Annette Muller-Taubenberger, Wolfram Schufer, and Gunther Gerisch Journal Biol. Chem., in press Abstract The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoidem cells. On the basis of mass spectrometric data, hisactophilin from the cytosol was analysed in parallel with hisactophilin extracted from a membrane fraction. None of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino-acid sequence of the protein, suggesting posttranslational modification. ES-MS and GC-MS of tryptic fragments separated by reversed-phase HPLC identified the most hydrophobic peptide as a myristylated fragment from the N-terminus of hisactophilin. From combination of the analytical data obtained, it is concluded that all hisactophilin in D. discoideum cells is N-terminally modified by myristylation. By reversed-phase HPLC, the D. discoideum hisactophilin could be separated into two isoforms which were recovered from both the cytosolic and membrane fraction. These isoforms, Hs I and Hs II, are distinguished by amino-acid sequence. Whereas the masses of Hs I fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristylation considered), Hs II is another protein distinguished from Hs I by several amino-acid exchanges. Hs I and Hs II can form homo- and heterodimers by disulfide bridges. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoidem, which may regulate the interaction of hisactophilin with the plasma membrane. ----------------------------------------------------------------------- [End CSM News, volume 3, number 19]