Dicty News Electronic Edition Volume 13, number 5 August 21, 1999 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 ============= The Regulative Capacity of Prespore Amoebae as Demonstrated by Fluorescence Activated Cell Sorting (FACS) and GFP Brian M. Nadin, Caroline S. Mah, James R. Scharff, and David I. Ratner Department of Biology, Amherst College, Amherst, MA, USA, 01002. Developmental Biology, in press. The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to re-establish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic micro-dissection work of K. Raper. The discovery of anterior like cells (ALCs) in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To re-examine this question, we performed fluorescence activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99.5%. Sorted GFP+ cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of "sham-sorted" (permissively gated, mixed GFP+ and GFP-) amoebae. This result confirms the developmental totipotency of prespore amoebae. ---------------------------------------------------------------------------- Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum transfer RNA genes Karol Szafranski (1), Gernot Glvckner (1), Theodor Dingermann (2), Karola Dannat (2), Angelika A. Noegel (3), Ludwig Eichinger (3), Andri Rosenthal (1) and Thomas Winckler (2) (1) Institut f|r Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745 Jena, Germany (2) Institut f|r Pharmazeutische Biologie, Universitdt Frankfurt/M. (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt/M, Germany (3) Institut f|r Biochemie I, Universitdt Kvln, Joseph-Stelzmann-Strasse 52, D-50931 Kvln, Germany Mol. Gen. Genet., in press Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream and Tdd-3 elements downstream of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are principle landmarks for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a coevolution process allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome carrying short intergenic DNA sequences. A new nomenclature is introduced to address all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5' and 3' of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C. ---------------------------------------------------------------------------- Structural Basis for Dimerization of the Dictyostelium Gelation Factor (ABP120) Rod Airlie J. McCoy 1, Paola Fucini 2,3, Angelika A. Noegel 2,4 and Murray Stewart1 1. MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, U.K. 2. Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, D-82152 Martinsried bei München, Germany. 3. Present address: New Chemistry Laboratories, South Parks Rd., Oxford OX1 3QT, U.K. 4. Institut für Biochemie I, Medizinische Einrichtungen der Universität zu Köln, Joseph-Stelzmann-Straße 52, D-50931 Köln, Germany. Nature Struct. Biol., in press. ABSTRACT Gelation factor (ABP120) is one of the principal actin-crosslinking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue sequence repeats that have an Ig fold. The ability to dimerize is crucial to the actin crosslinking function of gelation factor and is mediated by the rod in which the two chains are arranged antiparallel. We report the 2.2 Å resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, with domains 1-5 not paired with domains from the other chain. This information allows construction of an atomic model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact. ---------------------------------------------------------------------------- A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization Manabu Nakagawa1, Akiko A. Oohata2, Hiromasa Tojo3 and Shigeru Fujii1 1 Laboratory of Chemistry, Kansai Medical University, 18-89 Uyama-higashi, Hirakata, 573-1136, Japan 2 Laboratory of Biology, Kansai Medical University, 18-89 Uyama-higashi, Hirakata, 573-1136, Japan 3 Department of Molecular Physiological Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, 565-0871, Japan Biochemical Journal, in press Under starvation conditions, amoebae of Dictyostelium discoideum aggregate to form multicellular masses: the aggregates are then initiated to differentiate. We have reported previously that a signal substance exists in conditioned medium of D. discoideum, and we named it prespore-cell-inducing factor (psi factor) [Oohata, Nakagawa, Tasaka, and Fujii (1997) Development 124, 2781-2787]. The factor can induce isolated amoebae to differentiate into prespore cells. Moreover, we suggested that it caused not only cell differentiation but also cell division. In the present study, we have purified psi factor from the conditioned medium and characterized it. The purified psi factor induced both prespore cell differentiation and cell division of prespore cells. Its apparent molecular mass was 180 kDa by gel filtration and 106 kDa by SDS/PAGE. Based on these results, psi factor exists as a dimer in normal conditions. Periodic acid/Schiff staining showed that psi factor was a glycoprotein. It was ascertained by Edman degradation that psi factor is blocked at the N-terminal. Treatment with pyroglutamate aminopeptidase removed the N-terminal block and allowed determination of the amino-acid sequence of psi factor. Moreover, three internal amino-acid sequences were determined in limited proteolysis experiments using trypsin and endoproteinase Lys-C. The homology search for these sequences supports the fact that psi factor is a novel differentiation factor. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 5]