Dicty News Electronic Edition Volume 23, number 9 september 10, 2004 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Disruption of the NCS-1/frequenin-related ncsA gene in Dictyostelium discoideum accelerates development Barrie Coukell*, Anne Cameron, Stephen Perusini1, Katharine Shim Department of Biology, York University,4700 Keele St., Toronto, ON, M3J 1P3, CANADA Develop. Growth Differ., in press To learn more about the function of intracellular Ca2+ in Dictyostelium discoideum, we searched databases for sequences encoding potential members of the neuronal calcium sensor (NCS) family of Ca2+-binding proteins. As a result, genes for five new putative Ca2+-binding proteins were identified. Based on amino acid sequence alignments and phylogenetic analyses, one of these genes (ncsA) was determined to be closely related to NCS-1/frequenin genes in other organisms. The protein product of ncsA (NcsA) binds 45Ca2+ and exhibits a dramatic gel mobility shift in the presence of Ca2+, suggesting that it is a Ca2+ sensor. ncsA-null cells grow normally in axenic culture. However, on bacterial lawns, the ncsA-null clones expand slowly and development begins prematurely within the plaques. In larger clones, ncsA-null cells form narrow growth zones with evenly spaced aggregrates along the inner edge, and closely packed fruiting bodies. An analysis of intracellular cAMP levels, developmental timing on PBS agar, and stage-specific gene expression indicate that development of ncsA-null cells is accelerated by 3-4 h. Together, these results suggest that NcsA might function in Dictyostelium to prevent cells from entering development prematurely in the presence of environmental nutrients. Submitted by: Barrie Coukell [bcoukell@yorku.ca] ----------------------------------------------------------------------------- Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells Nao Shimada1, Toshinari Maruo2, Mineko Maeda2, Hideko Urushihara3 and Takefumi Kawata1, 4 1 Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan 2Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572 4 Corresponding author E-mail: tkawata@bio.sci.toho-u.ac.jp Differentiation, in press Abstract Dd-STATa, a Dictyostelium homologue of the metazoan STATs (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homologue of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determine. As expected from the behaviour of the endogenous aslA gene, the aslA:lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior; the pstAB core. During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk the aslA gene comes to be expressed in cells of the upper and lower cups; structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the prestalk regions of slugs and the stalks of culminants. Thus this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa. Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] ----------------------------------------------------------------------------- Molecular structure of the rod domain of Dictyostelium filamin Grzegorz M. Popowicz, Rolf Mueller, Angelika A. Noegel, Michael Schleicher, Robert Huber and Tad A. Holak J. Mol. Biol, in press Dictyostelium discoideum filamin (ddFLN) is a two-chain F-actin crosslinking protein with an N-terminal actin-binding domain and a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin (Ig) fold. We report the 2.8 resolution crystal structure of a homodimer of rod repeats 4, 5 and 6. The two chains are arranged in an antiparallel fashion and form an elongated element, which is shortened, however, compared to a fully extended, linear configuration because the long axis of each Ig domain is arranged at an angle to the long axis of the rod. Same arrangement of repeats should also be present in the rod domain of human FLNa, much longer than Dictyostelium FLN, which forms a straight and long rigid structure able to crosslink F-actin chains over distances of more than 1000 . Submitted by: Michael Schleicher [schleicher@lrz.uni-muenchen.de] ============================================================================== [End Dicty News, volume 23, number 9]