Dicty News Electronic Edition Volume 25, number 8 October 14, 2005 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 ============= Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination. Steve J. Charette?, Sophie Cornillon, Pierre Cosson. Universite de Geneve, Centre Medical Universitaire, Departement de Physiologie Cellulaire et Metabolisme, 1 rue Michel Servet, CH-1211 Geneve 4, Switzerland. Journal of Biotechnology, in press. Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event. Submitted by: Steve Charette [charett2@etu.unige.ch] ----------------------------------------------------------------------------- Receptor Occupancy on an Ellipsoidal Cell in the Presence of a Point Source of a Chemoattractant Igor Weber Department of Molecular Biology, Ruder Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, Croatia Journal of Chemical Information and Modeling, in press Diffusion of a chemoattractant from a micropipet is routinely used to examine the different aspects of a cell’s chemotactic response. To quantify the effect of cell elongation on chemotactic sensitivity in the micropipet assay, the chemoattractant concentration at the cell plasma membrane was determined by solving the equation for diffusion from a point source in the presence of a prolate ellipsoid of varying eccentricity. The results show that cell elongation can significantly increase the difference in receptor occupancy betweennear and far cell ends and thereby enhance the sensitivity of chemotactic cells to shallow chemoattractant gradients. Submitted by: Igor Weber [iweber@irb.hr] ----------------------------------------------------------------------------- Cryoelectron Tomography: Implications for Actin Cytoskeleton Research Igor Weber Department of Molecular Biology, Ruder Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, Croatia Croatica Chemica Acta, in press Disclosing undistorted spatial organization of the actin cytoskeleton with molecular resolution is fundamental for understanding the cellular ultrastructure. Whereas important insights into the architecture of microfilament networks have been gained by negative staining, critical point drying, and freeze-fracturing methods, it is cryoelectron tomography that provides, for the first time, a comprehensive three-dimensional view of the intact actin cytoskeleton in situ. In particular, topological relationships such as microfilament three-dimensional proximity, angles at filament branching points, and modes of microfilament interaction with the plasma membrane can be visualized with an unprecedented accuracy using this technique. Further improvements are expected to bring the resolution into the realm of 2–3 nm, where automatic pattern-recognition methods can be applied to identify actin-binding complexes. Combining cryoelectron tomography with ultra-fine immunolabeling and high-resolution fluorescence microscopy will make it possible to correlate structural data on the nanometer scale with molecular specificity and dynamical information. Submitted by: Igor Weber [iweber@irb.hr] ============================================================================== [End Dicty News, volume 25, number 8]