Dicty News Electronic Edition Volume 16, number 8 April 21, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ================================ News from the Genome Project ================================ PRELIMINARY DIRECTORY OF DICTYOSTELIUM GENES WEB SITE: http://dicty.sdsc.edu/annotationdicty.html Negin Iranfar, Rolf Olsen and William F. Loomis University of California San Diego La Jolla Cal. 92093 and The Dictyostelium Genome Project* We have prepared a Preliminary Directory of Dictyostelium Genes present in the contigs generated at the Sanger Centre by using a trained version of the genefinder program HMMgene (Krogh, 2000). The Preliminary Directory can accessed at http://dicty.sdsc.edu/annotationdicty.html. The list of 6,700 genes can be downloaded as an Excel file for sorting and searching. Rapid links are provided to the sequences of specific genes and the complete set can be searched by Blast programs. Using a challenge set of 20 genes we find that HMMgene trained on Dictyostelium genes is significantly more effective (98.9% of codons) than TIGR's GlimmerM trained with malarial genes (87.5% of codons). Krogh, A. (2000). Using database matches with HMMGene for automated gene detection in Drosophila. Genome Res. 10: 523-528. *The Dictyostelium Genome Project : The Baylor Sequencing Center, Houston, Texas, (A. Kuspa and R. Gibbs) where sequencing is supported by the NIH; the Institute of Biochemistry, Cologne together with the Institute of Molecular Biotechnology, Jena, Germany (G. Glöckner , A. Rosenthal, L. Eichinger and A. Noegel) where sequencing is supported by the Deutsche Forschungsgemeinschaft (No 113/10-1 and 10-2).; and the EUDICT consortium supported by The European Union (M-A. Rajandream, D. Lawson and B. Barrell). ============== Abstracts ============== RasG regulates discoidin gene expression during Dictyostelium growth D.M. Secko1, M. Khosla1, P. Gaudet3,4, A. Tsang3,4,5, G.B. Spiegelman1,2 and G. Weeks1,2* Department of Microbiology and Immunology1 and Department of Medical Genetics2, University of British Columbia, 6174 University Blvd., Vancouver, British Columbia, V6T 1Z3, Canada Department of Chemistry and Biochemistry3, Centre of Structural and Functional Genomics4, Department of Biology5, Concordia University, 1455 de Maisonneuve Blvd. W. Montreal, Quebec, H3G 1M8, Canada * To whom correspondence and reprint requests should be addressed. Fax: (604) 822-6041. Email: gweeks@unixg.ubc.ca. Exp. Cell Research, in press ABSTRACT When a pVEII-rasG(G12T) transformant was grown in HL5 media containing no folate, discoidin and RasG levels increased and then rapidly declined while cells were in the early stages of exponential growth, a result that contrasted with the much slower decline of discoidin during the growth of Ax-2. The levels of discoidin in Ax-2 were considerably higher than the levels in the pVEII-rasG(G12T) transformant. These results suggested the possibility that RasG(G12T) represses discoidin gene expression. When the rasG(G12T) gene was placed under the control the ribonucleotide reductase (rnrB) promoter, RasG(G12T) was rapidly generated upon exposure to 10 mM methyl methanesulfonate (MMS) and this was accompanied by an equally rapid decrease in discoidin mRNA levels, independently confirming that RasG(G12T) represses discoidin gene expression. Despite the repression of discoidin expression by activated RasG, rasG null cells contained decreased levels of discoidin, indicating that endogenous RasG is essential for optimum discoidin expression. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter. ----------------------------------------------------------------------------- Functional and regulatory analysis of the Dictyostelium G-box binding factor Jason M. Brown and Richard A. Firtel Developmental Biology, in press. ABSTRACT The Dictyostelium discoidium G-box Binding Factor (GBF) is required for the induction of known postaggregative and cell-type-specific genes. gbf null cells undergo developmental arrest at the loose-mound stage due to the absence of GBF-targeted gene transcription. GBF-mediated gene expression is activated by stimulation of cell-surface, seven-span cAMP receptors, but this activation is independent of heterotrimeric G-proteins. To further characterize GBF, we assayed a series of GBF mutants for their ability to bind a G-box in vitro and to complement the gbf null phenotype. In vitro DNA-binding activity resides in the central portion of the protein, which contains two predicted zinc fingers. However, in vivo GBF function requires only one intact zinc finger. In addition, expression of some GBF mutants results in a partial complementation phenotype, suggesting that these mutants are hypomorphic alleles. We used a 2.4 kb GBF-promoter fragment to examine the regulation of GBF expression. GBF promoter-reporter studies confirmed the previous finding that GBF transcription is induced by continuous, micromolar extracellular cAMP. We also show that, like the activation of GBF-regulated transcription, the induction of GBF expression requires cell-surface cAMP receptors, but not heterotrimeric G-proteins. Finally, reporter studies demonstrated that induction of GBF-promoter-regulated expression does not require the presence of GBF protein, indicating that GBF expression is not regulated by a positive autoregulatory loop. ----------------------------------------------------------------------------- FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum Takahiro Morio (1) , Hiroo Yasukawa (2), Hideko Urushihara (1), Tamao Saito (3), Hiroshi Ochiai (3), Ikuo Takeuchi (4), Mineko Maeda (5) and Yoshimasa Tanaka (1) (1) Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. (2) Molecular Biology Group, Faculty of Engineering, Toyama University, Toyama 930-8555, Japan. (3) Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. (4) Novartis Foundation for the Promotion of Science, Takarazuka, Hyogo 665-0042, Japan. (5) Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan. BBA, in press. Summary We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells. ----------------------------------------------------------------------------- Total tetra knockout of GP138 multigene family implicated in cell interactions in Dictyostelium discoideum Key words: sexual cell fusion, multigene, gene disruption Toshihiro Hata, Mina Takahashi, Yoshimasa Tanaka, and Hideko Urushihara * Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, 305-8572 Japan * Address correspondence to: Hideko Urushihara, Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, 305-8572 Japan Gene, in press. ABSTRACT The cellular slime mold Dictyostelium discoideum reproduces sexually under submerged and dark conditions. A cell surface glycoprotein gp138 has been identified as a target molecule for cell fusion-blocking antibodies, and is considered to be indispensable for the sexual cell fusion in this organism. Currently, four isoforms of gp138, DdFRP1a, DdFRP1b, DdFRP2, and DdFRP3, are known. Genes encoding the latter three isoforms, GP138C, GP138A, and GP138B, have been isolated, comprising a GP138 multigene family. Here we isolated the fourth GP138 gene, GP138D, encoding DdFRP1a. These GP138 genes were found to cluster in a tandem array on chromosome 5, being bordered by two GP138-like sequences highly homologous to them but truncated. To clarify functional relationships among the GP138 family members, the entire GP138 region was deleted by a single knockout. Northern hybridization and western immuno-blotting analyses confirmed complete losses of GP138 mRNA and DdFRPs in the knockout strains, indicating that there are no more GP138 genes. Unexpectedly, however, the GP138-null mutants were fully potent for both sexual cell fusion and subsequent development. In addition, the original fusion-blocking antibodies detected a cell surface protein of close electrophoretic mobility to gp138 in the knockouts, suggesting the possibility that the actual target molecule of the fusion-blocking antibodies was not DdFRPs but this unidentified component. Since GP138-null mutants exhibited no obvious defects either in growth or asexual development, the real function of the GP138 family is unknown. Nevertheless, the expression levels of other developmental genes such as acaA, csaA, cotA-C, and spiA appeared to be altered in the GP138-null mutants. Therefore, it seems to have a non-critical but some role(s) during asexual development. ----------------------------------------------------------------------------- cAMP receptor affinity controls wave dynamics, geometry and morphogenesis in Dictyostelium Dirk Dormann, Ji-Yun Kim, Peter N Devreotes and Cornelis J.Weijer Journal of Cell Science (2001), in press Serpentine, G protein coupled, cAMP receptors are key components in the detection and relay of the extracellular cAMP waves that control chemotactic cell movement during Dictyostelium development. During development the cells sequentially express four closely related cAMP receptors of decreasing affinity. In this study we investigate the effect of cAMP receptor type and affinity on the dynamics of cell-cell signalling in-vivo, by measuring the dynamics of wave initiation and propagation in a variety of cAMP receptor mutants. We find that receptor affinity controls the frequency of wave initiation, while it does not determine wave propagation velocity, thus resulting in dramatic changes in wave geometry. In the limiting case the affinity of the receptor is so low that waves can still be initiated but no stable centres form and as a result the cells cannot aggregate. In mounds expression of low affinity receptors results in very slow concentric waves instead of the normally observed multi-armed spiral waves. Under these conditions there is no rotational cell movement and the hemispherical mounds cannot transform into slugs. These results highlight the importance of receptor number and affinity in the proper control of cell-cell signalling dynamics required for the successful completion of development. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 8]