Dicty News Electronic Edition Volume 15, number 1 July 8, 2000 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 ============== Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells. Mieke Blaauw, Maarten H.K. Linskens and Peter J.M. van Haastert Cell Engineering Facility GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Gene, in press. Abstract We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTAs*). The second component is an extra-chromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promotor contains a tetracycline-responsive element, which is the binding site for tTAs*. Tetracycline prevents tTAs* from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTAs* binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene. ---------------------------------------------------------------------------- Mutant Rac1B Expression in Dictyostelium: Effects on Morphology, Growth, Endocytosis, Development, and the Actin Cytoskeleton Stephen J. Palmieri1, Thomas Nebl1*, Robert K. Pope1*, David J.Seastone2, Enkyung Lee3, Edward H. Hinchcliffe1, Greenfield Sluder1, David Knecht3, James Cardelli2, and Elizabeth J. Luna1 1Department of Cell Biology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, MA 01605 2Department of Microbiology and Immunology, Feist-Weiller Cancer Center, Louisiana State University Medical Center, Shreveport, LA 71130 3Department of Molecular and Cellular Biology, University of Connecticut, Storrs, CT 06269 *T. Nebl and R. K. Pope contributed equally to this work. Cell Motility and the Cytoskeleton, in press. SUMMARY Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12ÆV or Q61ÆL) or a dominant negative mutant (T17ÆN) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally- expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations. ---------------------------------------------------------------------------- Spatial and Temporal Expression of a Polysphondylium Spore-specific Gene. Keqin Y. Gregg & Edward C. Cox 333 Moffett Laboratories, Department of Molecular Biology, University, Princeton, New Jersey 08544 Developmental Biology, in press. SUMMARY In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106 bp fragment between positions -246 and –352 of the upstream coding sequence. This fragment can be further divided into a promoter proximal and promoter distal PRE and a 29 bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates, and that cell sorting plays little if any role in this process. ---------------------------------------------------------------------------- Pictures in Cell Biology, The Dictyostelium fruiting body – a stucture of cells and cellulose Supriya Srinivasan, Hannah Alexander and Stephen Alexander Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 Accepted, Trends in Cell Biology, August 2000 The Dictyostelium fruiting body is featured along with a descritption of its biology. ---------------------------------------------------------------------------- Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium Guochun Li 1, Hannah Alexander1, Natalie Schneider2 and Stephen Alexander 1 1 Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 and 2 Max-Planck Institut fur Biochemie, D-82152 Martinsried, Germany Accepted Microbiology ABSTRACT The efficacy of the widely used chemotherapeutic drug cisplatin is limited by the occurrence of drug-resistant tumor cells. To fully exploit the potential of this drug in cancer therapy, it is imperative to understand the molecular basis of cisplatin resistance. Using an insertional mutagenesis technique in cells of Dictyostelium discoideum, we have identified six genes which are involved in cisplatin resistance. None of these genes have been previously linked to resistance to this drug. Several of these genes encode proteins that are involved in signal transduction pathways which regulate cell death, cell proliferation or gene regulation. The resistance of these mutant strains is specific for cisplatin since deletion of these genes does not confer resistance to other DNA damaging agents. Significantly, the disruption of three of these genes, encoding the sphingosine-1-phosphate lyase, the regA cAMP phosphodiesterase and a phosphatidylinositol 4-phosphate 5-kinase, also results in abnormalities in the multicellular development of this organism, although there is no change in the rate of mitotic cell growth. This study identified previously unsuspected molecular pathways which function in the cellular response to cisplatin and are required for normal morphogenesis, and underscores the complexity of the cellular response to cisplatin. These pathways provide potential targets for modulating the response to this important drug. ---------------------------------------------------------------------------- Green Fluorescent Protein (GFP) and Epitope Tag Fusion Vectors for Dictyostelium discoideum. Stephanie Levi, Mark Polyakov, and Thomas T. Egelhoff* Department of Physiology and Biophysics Case Western Reserve School of Medicine Cleveland, OH 44106-4970 USA IN PRESS, PLASMID We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed in a G418-resistance vector allowing either trans-activated Ddp2-based extrachromosomal replication, or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP expressing cells were analyzed by fluorescent microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 1]