Dicty News Electronic Edition Volume 21, number 3 July 25, 2003 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. ============= Abstracts ============= Influence of medium composition on growth behaviour of Dictyostelium discoideum for cultivation on axenic media Miriam Stephan, Usama Beshay, Karl Friehs, and Erwin Flaschel Bielefeld University Faculty of Technology D-33594 Bielefeld, Germany Process Biochemistry, in press The social amoeba D. discoideum represents an attractive host organism for the production of heterologous proteins. However, its application is seriously affected by slow growth rates as well as low maximal cell densities in the presence of axenic (liquid) media. Starting with standard complex media the influence of certain medium components is investigated. Thus, the kind and concentration of carbohydrates, the concentration of salt and ammonia as well as the supplementation of conditioned media are being varied. These studies are performed by following cell growth in batch experiments over the whole growth cycle into the decline phase to obtain information about growth rates as well as maximal cell densities. Only maltose, glucose and a-trehalose are metabolized with appreciable rates. The concentration of ammonia produced correlates inversely with the concentration of carbohydrates metabolized. Under standard conditions ammonia reaches concentrations of about 50 mM, most of which being produced during stationary phase. Ionic strength and the addition of ammonia affect the growth rate as well as the maximal cell density. Ammonia can not be accused to limit the maximal cell density. Finally, it is shown that D. discoideum can be cultivated in normal stirred bioreactors. A semi-empirical model is discussed for the description of the growth behaviour. Submitted by: Erwin Flaschel [efl@fermtech.techfak.uni-bielefeld.de] ----------------------------------------------------------------------------- Improvement of a synthetic medium for Dictyostelium discoideum Sang-In Han, Karl Friehs and Erwin Flaschel Bielefeld University Faculty of Technology D-33594 Bielefeld, Germany Process Biochemistry, in press D. discoideum is of considerable interest as an expression system for the production of proteins of high value. The cultivation of this social amoeba is not as easy as with other common microbial expression systems. Wildtype strains grow on bacteria. Mutant strains growing on axenic media reach cell densities of 1-2á10^7 mL- 1 when cultivated in commonly used complex media. A totally synthetic medium formulated by Franke and Kessin in 1977 has become popular and allows cell densities of about 3á10^7 mL-1 to be obtained. This medium (FM) is being improved mainly on the basis of the analysis of limitations with respect to amino acids. With this improved synthetic medium (SIH) cell densities in the order of 5- 6á10^7 mL-1 have been achieved. Submitted by: Erwin Flaschel [efl@fermtech.techfak.uni-bielefeld.de] ----------------------------------------------------------------------------- Cultivation of Dictyostelium discoideum on an improved synthetic medium in a conventional bioreactor Sang-In Han, Karl Friehs and Erwin Flaschel Bielefeld University Faculty of Technology D-33594 Bielefeld, Germany Process Biochemistry, in press An improved fully synthetic medium has been presented recently based on the synthetic medium formulated by Franke and Kessin in 1977. This novel medium was called SIH instead of the classical FM medium. The improved medium leads to even higher cell densities, a more balanced uptake of amino acids and a better utilisation of glucose. However, the growth behaviour had only been assessed during shake-flask cultivation. Here it is shown that D. discoideum can be cultivated in conventional stirred tank-type bioreactors, if the operating conditions take care about the shear sensitivity of the cells. A batch as well as a fed-batch cultivation show that mass cultivation of D. discoideum can be achieved during growth on synthetic media. Submitted by: Erwin Flaschel [efl@fermtech.techfak.uni-bielefeld.de] ----------------------------------------------------------------------------- A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle Natalie Schneider 1,2, Igor Weber 2,3, Jan Faix 1, Josef Prassler 2,Ê Annette Mueller-Taubenberger 2, Jana Koehler 2, Emmanuel Burghardt 2,Ê Guenther Gerisch 2 and Gerard Marriott 1 1 University of Wisconsin-Madison, Department of Physiology, Madison, WI 53706, USA; 2 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany; 3 Rudjer-Boskovic-Institut, Bijenicka cesta 54, 10000 Zagreb, Croatia. Cell Motility and the Cytoskeleton, in press. DdLimE regulates cell motility and cytokinesis in Dictyostelium. To specify its function, we generated knock-out mutants and analyzed mitosis by marking the mitotic apparatus with GFP-a-tubulin. Characteristic of DdLimE-null cells is a late reversal of cytokinesis caused by backward movement of the incipient daughter cells. This process of Òretro-cytokinesisÓ is accompanied by a delay in disassembly of the mitotic spindle. The length of interphase microtubules is increased and their depolymerization at prophase is impaired. These data indicate that DdLimE links the cortical actin network, where it is located, to the microtubule system, whose dynamics it regulates. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] ----------------------------------------------------------------------------- **************************************************************************** NOTE: The biological materials necessary to use this method are being lodged with Dr Jacob Franke's Dictyostelium Stock Centre **************************************************************************** Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development Tomoaki Abe, Judith Langenick and Jeffrey G. Williams* School of Life Sciences University of Dundee Wellcome Trust Biocentre Dow Street DUNDEE, DD1 5EH, UK (Gene disruption, transposition, Dictyostelium, qkgA gene) We describe a rapid method for creating Dictyostelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topo-isomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bsr) cassette linked to a bacterial tetracycline resistance (tetr) cassette. After transposition the plasmid DNA is transformed into E. coli and clones in which the bsr-tetr cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictystelium kinases, GbpC and pats1. Like them it contains a Leucine Rich Repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures. Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] =============================================================================== [End Dicty News, volume 21, number 3]