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dictyNews Volume 21 Number 03
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.
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Abstracts
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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]
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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]
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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]
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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]
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****************************************************************************
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]
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[End Dicty News, volume 21, number 3]
