CSM News Electronic Edition Volume 6, number 5 February 24, 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" =========== Abstracts =========== Mutagenesis and gene identification in Dictyostelium by shotgun antisense Timothy P. Spann, Debra A. Brock, David F. Lindsey, Salli A. Wood, and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 PNAS, in press ABSTRACT We have developed a mutagenesis technique that uses antisense cDNA to identify genes required for development in Dictyostelium discoideum. We transformed Dictyostelium cells with a cDNA library made from the mRNA of vegetative and developing cells. The cDNA was cloned in an antisense orientation immediately downstream of a vegetative promoter, so that in transformed cells the promoter will drive the synthesis of an antisense RNA transcript. We find that individual transformants typically contain one or occasionally two antisense cDNAs. Using this mutagenesis technique, we have generated mutants which either fail to aggregate, aggregate but fail to form fruiting bodies, or aggregate but form abnormal fruiting bodies. The individual cDNA molecules from the mutants were identified and cloned using PCR. Initial sequence analysis of the PCR products from 35 mutants has identified six novel Dictyostelium genes, each from a transformant with one antisense cDNA. When the PCR-isolated antisense cDNAs were ligated into the antisense vector and the resulting constructs transformed into cells, the phenotypes of the transformed cells matched those of the original mutants from which each cDNA was obtained. We made homologous recombination gene disruption transformants for three of the novel genes, in each case generating mutants with phenotypes indistinguishable from those of the original antisense transformants. Shotgun antisense thus is a rapid way to identify genes in Dictyostelium and possibly other organisms. ---------------------------------------------------------------------- Cyclic AMP dependent protein kinase and prestalk-cell gene expression in Dictyostelium. Fabiana Horn and Julian D. Gross Department of Biochemistry, University of Oxford South Parks Road,Oxford OX1 3QU, U.K. FEMS Microbiol. Letters, in press. ABSTRACT Cyclic AMP-dependent protein kinase (PKA) is required for aggregation of starved amoebae during development of Dictyostelium discoideum as well as for prespore gene expression and for maturation of stalk cells and spores. It has not been clear until now whether it plays any role in the stalk pathway prior to the final maturation of stalk cells. We have examined stalk cell formation and prestalk cell gene expression in a mutant of Dictyostelium that has an inactive PKA regulatory subunit and therefore has unrestrained catalytic activity [1]. We show that when mutant amoebae are incubated under buffer in the absence of cAMP they accumulate transcripts of the prestalk-cell-specific ecmA gene and form stalk cells, whereas the parental cells neither accumulate ecmA transcripts nor form stalk cells. These findings indicate that constitutive PKA activity renders cells able to express prestalk-cell-specific genes under conditions where wild type cells cannot do so, and hence that PKA is probably implicated in prestalk gene expression during normal development. ------------------------------------------------------------------- MULTIPLE ROLES OF THE NOVEL PROTEIN-TYROSINE PHOSPHATASE PTP3 DURING DICTYOSTELIUM GROWTH AND DEVELOPMENT Marianne Gamper, Peter K. Howard, Tony Hunter, and Richard A. Firtel Molecular and Cellular Biology, in press. ABSTRACT PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic PTP domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell-types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys->Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of variable size. Anti-phosphotyrosine (pTyr) Western blot analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with non-nutrient buffer led to reciprocal changes in the pattern of several pTyr proteins, including a protein of ~130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested p130 is a potential substrate of PTP3. A transient post-translational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data shown strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2. -------------------------------------------------------------------- MOLECULAR GENETICS OF SIGANL TRANSDUCTION IN DICTYOSTELIUM Carole A. Parent and Peter N. Devreotes Annual Review of Biochemistry, in press. ABSTRACT In conditions of starvation, the free living amoebae of Dictyostelium enter a developmental program - the cells aggregate by chemotaxis to form a multicellular structure that undergoes morphogenesis and cell-type differentiation. These processes are mediated by a family of cell surface cAMP receptors (cARs) that act on a specific heterotrimeric G protein to stimulate actin polymerization, activation of adenylyl and guanylyl cyclases, and a host of other responses. Most of the components in these pathways have mammalian counterparts. The accessible genetics of this unicellular organism facilitate structure-function analysis and enable the discovery of novel genes involved in the regulation of these important pathways. ---------------------------------------------------------------------- Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation Takefumi Kawata*, Anne Early* and Jeffrey Williams+ MRC Laboratory of Molecular Cell Biology and Dept of Biology University College London, Gower St, London WC1E 6BT, U.K. *Joint first authors EMBO J., in press. ABSTRACT The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by DIF, a low molecular weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered. ------------------------------------------------------------------- Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular Slime Mold, Polysphondylium pallidum Ochiai Hiroshi, Hata Katsusuke, Saito Tamao, Funamoto Satoru, and Nakata Nobuyuki Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060 Japan Plant Cell Physiology, in press. SUMMARY The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhesion. The major component of them has been identified as a glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to as gp64). We found that a substantial amount of the gp64 run as dimer, when gp64 was dissolved in SDS-sample buffer without 2-mercaptoethanol and then subjected to electrophoresis. The occurrence of a homophilic dimer was demonstrated by analyzing the dimer-like band on a gel for its amino acid sequence and amino acid composition. The dimer-like band also was analyzed by three sorts of monoclonal antibodies, two of which recognize respectively a conformational epitope and a denatured epitope of the protein moiety of gp64. The data indicate that the native conformation of gp64 is necessary for dimer formation. The cellular slime mold Polysphondylium pallidum is like a related species, Dictyostelium discoideum, whose cells migrate in response to a chemoattractant secreted from center cells and finally form a multicellular aggregate (Gerisch et al. 1980). When cells of the two species are mixed and plated onto a solid agar surface, they sort out into separate aggregates not only because of differences in chemoattractants but also because of species-specific cell-cell adhesion (Gerisch et al. 1980). D. discoideum has two types of adhesion systems, named the contact site A and the contact site B (Beug et al. 1973). The contact site A was identified as a glycoprotein with a molecular mass of 80 kDa (Mueller et al ., 1979) and has been well studied. Its cDNA sequence (Noegel et al., 1986), an adhesion site (Kamboj et al. 1989) and a glycosyl phosphatidylinositol anchor (Stadler et al. 1989) have been reported. The contact site B also has been preliminary identified as a gp120 (Chadwick et al. 1984), or a gp 24 (Knecht et al. 1987). In the related species P. pallidum two EDTA-resistant types of cell adhesion have been described (Bozzaro and Gerisch, 1978). One is a minor component which begins to appear at aggregation stage and the other is a major component expressed already during growth phase and persisting until later stages. The major component was identified as s glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to as gp64) (Bozzaro et al. 1981). Monoclonal antibodies against purified gp64 completely blocked cell-cell adhesion of aggregation-competent P. pallidum cells and this blocking activity was neutralized with L-fucose (Toda et al 1984). This result indicates that the carbohydrate moiety of gp64 is involved in cell-cell adhesion. Recently, by subjecting purified gp64 to electrophoresis, we recognized the presence of a dimer-like molecule on SDS-gel. Bozzaro et al. (1981) also noticed the same thing, but they did not analyze the nature of the dimer though they suggested that gp64 has a tendency to dimerize. We show here that the dimer-like product is a homophilic dimer of gp64 molecules and that the monomers might have a rather strong affinity to interact to each other. ---------------------------------------------------------------------- Antisense RNA Inactivation of Gene Expression of a Cell-Cell Adhesion Protein (gp64) in the cellular slime mold Polysphondylium pallidum Satoru Funamoto and Hiroshi Ochiai Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060 Japan J. Cell Sci. in press SUMMARY The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into the cells, and the resultant was characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hours starved L3mc2 cells remained considerably dissociated from each other after 5-minutes gyrating, although then gradually increased aggregation by 50% during a further 55-minutes gyrating, in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report which an antisense RNA technique was successfully applied to Polysphondylium. --------------------------------------------------------------------- Identification of a novel all-cis-5,9,12-heptadecatrienoic acid in the cellular slime mold Polysphondylium pallidum Tamao Saito and Hiroshi Ochiai Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060 Japan Lipids, in press. Summary All-cis-5,9,12-heptadecatrienoic acid, was identified in the cellular slime mold Polysphondylium pallidum. The structural elucidation was accomplished by capillary GC, argentation TLC and GC/MS. This fatty acid has not been reported previously. ----------------------------------------------------------------------- [End CSM News, volume 6, number 5]