Dicty News Electronic Edition Volume 12, number 5 February 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" ================================================================== REPORT ON THE NIH MEETING CONCERNING NON-MAMMALIAN MODEL SYSTEMS ================================================================== Rich Kessin, Bill Loomis, Carole Parent About 120 scientists and NIH administrators met on Tuesday and Wednesday, February 16th and 17th, at the Natcher Center in Bethesda to consider new initiatives that could support futher studies in non-mammalian model systems. Harold Varmus, Head of the NIH, opened the sessions by pointing out that the Institutes at NIH had been set up over the last 50 years on the basis of perceived needs for studies in specific organs or diseases. He felt that basic biological studies, especially in model organisms, fit uneasily in this structure and that NIH wide policies could be better adapted to present needs. This meeting focused on the needs of the communities that work with yeast, worms, flies, fish, frogs, and additional non-mammalian systems. Ira Herskowitz outlined the strengths of S. cereviasiae; Martin Chalfie described the advantages of C.elegans; Gerry Rubin went over D. melanogaster; Will Talbot talked about zebrafish and Marc Kirschner elequently emphasized the fusion of biochemistry and genetics in Xenopus. The organisms that were under consideration as additional model systems included Chlamydomonas, Tetrahymena, Dictyostelium, S. pombe, Neurosporra, Aplysia, sea urchins, Fugu and chickens. In a 10 minute presentation we reminded the participants of the power of microbial and molecular genetics techniques in Dictyostelium as well as the excellent cytology and biochemistry possible with this organism. The EST Project and the initiation of the Genome Project for Dictyostelium puts the system in a strong position for further utilization. The general consensus appeared to be that studies in Dictyostelium are ripe for major expansion. Strong arguments were also made for other systems including: Tetrahymena by Martin Gorovsky; Neurosporra by Bob Metzenberg; Aplysia by Eric Kandel; sea urchins by Eric Davidson, and Fugu by Sydney Brenner. Increased support by the NIH for studies in several of these systems appears likely. White papers outlining the needs of the community were prepared for the five established model systems (yeast, worms, flies, fish and frogs) as well as Dictyostelium and Tetrahymena. We emphasized the need for addition funds to complete the genome sequence, annotate it, enhance DictyDB, and establish a stock center. We also pointed out the need for strong support of genome array technology for expression analyses and future support for global gene knock-out studies. We estimated that a total of 4.6 million dollars additonal support would fullfill these essential needs. It was an intense, well-structured meeting in which excellent science as well as policy matters were discussed. We feel that the wider community gained an appreciation of the strengths of Dictyostelium as a model system as well as the strengths of the Dicty community. We are hopefull that we will see added NIH support in the near future. ============== Abstracts ============== Variables controlling the expression level of exogenous genes in Dictyostelium Ka Ming Pang, Michael A. Lynes, David A. Knecht University of Connecticut, Department of Molecular and Cell Biology Storrs, CT 06269, USA Plasmid, in press. Abstract Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescence protein (GFP) gene from vectors containing each of the drug resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin resistant cassette (pDNeoGFP), followed by the hygromycin resistant cassette (pDHygGFP) and the blasticidin resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate co-precipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP. ---------------------------------------------------------------------------- Isolation of spermidine synthase gene (spsA) of Dictyostelium discoideum1 Kunde Guo, Wen-Tsan Chang and Peter C. Newell Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK 1 The nucleotide sequence data has been submitted to Genbank under the accession number AF069757. Accepted: Biochimica et Biophysica Acta Summary Polyamines such as putrescine (1,4-diaminobutane), spermidine and spermine have been implicated in a wide variety of essential biological reactions including the biosynthesis of DNA and RNA in all cells from those of man to E. coli. In Dictyostelium, the major polyamines that have been reported in amoebae are spermidine, putrescine and 1,3-diaminopropane. In polyamine biosynthesis, the enzyme spermidine synthase converts putrescine to spermidine by transfer of a propylamine group from decarboxylated S-adenosylmethionine to putrescine. Using the gene disruption technique of Restriction Enzyme Mediated Integration (REMI), we have isolated the spermidine synthase gene from Dictyostelium discoideum and have designated it spsA. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 hours of development. Sequencing of the genomic DNA and a full length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence was found to be highly conserved, with amino acid identities compared to human spermidine synthases of 59.5%, mouse: 61.3% and yeast: 58.1%. A null mutant of the spsA gene was unable to grow in the absence of exogenous spermidine and growth was proportional to the amount of spermidine added. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that had abnormally short stalks. ---------------------------------------------------------------------------- Regulation of Dictyostelium Protein tyrosine Phosphatase PTP3 through Osmotic Shock and Stress Stimulation and Identification of pp130 as a PTP3 Substrate Marianne Gamper, Eugene Kim, Peter K. Howard, Hui Ma, Tony Hunter, and Richard A. Firtel, J. Biol. Chem., in press. ABSTRACT Osmotic shock and growth-medium stimulation of Dictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the ~130 kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein tyrosine phosphatase PTP3. This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 5]