CSM News Electronic Edition Volume 8, number 14 May 31, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========================== Dicty Researcher Database =========================== Also, please if you have not completed your entry in the Dicty Researcher Database on the Dicty web page or by following this link "http://apps.basic.northwestern.edu/dicty/dictyqbe.html" . =========== Abstracts =========== Chemotaxis to cAMP and Slug Migration in Dictyostelium Both Depend on MigA, a BTB Protein Ricardo Escalante, Deborah Wessels, David R. Soll and William F. Loomis Mol. Biol. Cell in press. Abstract Chemotaxis in natural aggregation territories as well as in a chamber with an imposed gradient of cAMP was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (REMI). We picked it because it formed small, misshapen fruiting bodies. Following isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full length migA gene and its upstream regulatory region, rescued the defects. The sequence of the full length gene revealed that it encodes a novel protein with a BTB domain near the N-terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. While migA-cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients and its absence results in defective chemotaxis and slug migration. --------------------------------------------------------------------- The cluA- mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria. Zhu, Q., Hulen, D., Liu, T., and Clarke, M. Proc. Natl. Acad. Sci. USA, in press ABSTRACT The clu A gene of Dictyostelium discoideum encodes a novel 150-kDa protein. Disruption of cluA results in clustering of mitochondria near the cell center. This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm. The mutant cell populations also exhibit an increased frequency of multinucelated cells, suggesting an impairment in cytokinesis. Both phenotypes are reversed by transformation of cluA- cells with a plasmid carrying a constitutively expressed cluA gene. The predicted sequence of the cluA gene product is homologous to sequences encoded by open reading frames in the genomes of S. cerevisiae and C. elegans, but not to any known protein. The only exception is a short region with some homology to the 42-residue imperfect repeat in the kinesin light chain, which probably function in protein-protein interaction. These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria. --------------------------------------------------------------------- Analysis of mRNA levels for the developmentally regulated prespore specific glutamine synthetase in Dictyostelium discoideum. Andrew J. Dunbar and John F. Wheldrake* Cooperative Research Centre for Tissue Growth and Repair, GPO Box 10065, Adelaide, Australia. *Flinders University of South Australia, Faculty of Science and Engineering, School of Biological Sciences. GPO Box 2100, Adelaide 5001, Australia. Development Growth and Differentiation, in press The enzyme glutamine synthetase (GS) of D. discoideum is developmentally regulated, preferentially localised in prespore cells and is likely to play an important role in controlling the levels of ammonia, a known morphogen, in this organism. To investigate further the regulation of GS we have isolated of a portion of the GS gene and used it as a probe to examine the changes in GS mRNA throughout development and the level of GS mRNA in the two precursor cell types. The amino acid sequence of the cloned DNA fragment we have isolated is highly homologous to other eukaryotic GS genes. DNA blot analysis demonstrates that the GS gene exists as a single copy in D. discoideum. Analysis of RNA indicates that there is a single 1.7 Kb GS transcript which increases during development to peak at the initial stages of culmination. Furthermore the GS mRNA is highly localised in prespore cells which is consistent with a proposed source-sink model for ammonia assimilation in this organism. --------------------------------------------------------------------- Effect of the glutamine synthetase inhibitor, methionine sulphoximine, on the growth and differentiation of Dictyostelium discoideum Andrew J. Dunbar and John F. Wheldrake* Cooperative Research Centre for Tissue Growth and Repair, GPO Box 10065, Adelaide, Australia. *Flinders University of South Australia, Faculty of Science and Engineering, School of Biological Sciences. GPO Box 2100, Adelaide 5001, Australia. FEMS Letters, in press To examine further the role of the enzyme glutamine synthetase (GS) in D. discoideum we report here the effects of a specific glutamine synthetase inhibitor, methionine sulphoximine (MSX), on the growth and differentiation of this organism. Vegetative AX3 cells grown in the presence of MSX did not complete culmination in the normal time but were blocked at the finger stage. In these cells GS activity was almost completely abolished. However, MSX did not affect the level of GS mRNA, suggesting that there is no link between GS activity and mRNA transcripition. Eventually GS activity reappeared and at the time culmination occurred. These results suggest that GS plays an important role in the assimilation of ammonia during the later stages of development in D. discoideum and that this assimilation is necessary for the completion of culmination. --------------------------------------------------------------------- [End CSM News, volume 8, number 14]