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dictyNews Volume 21 Number 18
Dicty News
Electronic Edition
Volume 21, number 18
December 12, 2003
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.
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Special Note to the Dictyostelium community
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For all of the dicty community preparing RNAi constructs:
In the article mentioned below we describe the difficulties we met
when attempting to purify fragments consisting of inverted-repeat
sequences of D. discoideum by using kit for isolation of DNA from
agarose gel. The DNA may be denaturated during the procedure and
reasociated into monomolecular hairpins.
Martin Prevorovsky, Frantisek Puta
The A/T rich inverted repeats are destabilized by chaotrope-containing
buffer during purification from agarose gel.
Biotechniques. 2003 Oct;35(4):698-700, 702
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Abstracts
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ADENYLYL CYCLASE G IS ACTIVATED BY AN INTRAMOLECULAR OSMOSENSOR
Shweta Saran and Pauline Schaap
School of Life Sciences, University of Dundee, UK
Mol. Biol. Cell, in press
Adenylyl cyclase G (ACG) is activated by high osmolality
and mediates inhibition of spore germination by this stress
factor. The catalytic domains of all eukaryote cyclases are
active as dimers and dimerization often mediates activation.
To investigate the role of dimerization in ACG activation, we
co-expressed ACG with an ACG construct that lacked the
catalytic domain (ACG)cat) and was driven by a UV-
inducible promoter. After UV-induction of ACG)cat, cAMP
production by ACG was strongly inhibited, but
osmostimulation was not reduced. Size-fractionation of native
ACG showed that dimers were formed between ACG
molecules and between ACG and ACG)cat. However, high
osmolality did not alter the dimer/monomer ratio. This
indicates that ACG activity requires dimerization via a region
outside the catalytic domain, but that dimer formation does not
mediate activation. To establish whether ACG required
auxiliary sensors for osmostimulation, we expressed ACG
cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP
production by ACG was similarly activated by high osmolality
as in Dictyostelium. This strongly suggests that the ACG
osmosensor is intramolecular, which would define ACG as the
first characterized primary osmosensor in eukaryotes.Ê
Submitted by: Pauline Schaap [p.schaap@dundee.ac.uk]
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Identification and phylogenetic analysis of Dictyostelium discoideum kinesin
proteins
Martin Kollmar and Gernot Gloeckner
BMC Genomics 2003, 4:47 (published 27 November 2003)
Background
Kinesins constitute a large superfamily of motor proteins in eukaryotic
cells. They perform diverse tasks such as vesicle and organelle transport
and chromosomal segregation in a microtubule- and ATP-dependent manner.
In recent years, the genomes of a number of eukaryotic organisms have
been completely sequenced. Subsequent studies revealed and classified
the full set of members of the kinesin superfamily expressed by these
organisms. For Dictyostelium discoideum, only five kinesin superfamily
proteins (Kif's) have already been reported.
Results
Here, we report the identification of thirteen kinesin genes exploiting
the information from the raw shotgun reads of the Dictyostelium
discoideum genome project. A phylogenetic tree of 390 kinesin motor
domain sequences was built, grouping the Dictyostelium kinesins into
nine subfamilies. According to known cellular functions or strong
homologies to kinesins of other organisms, four of the Dictyostelium
kinesins are involved in organelle transport, six are implicated in
cell division processes, two are predicted to perform multiple functions,
and one kinesin may be the founder of a new subclass.
Conclusion
This analysis of the Dictyostelium genome led to the identification of
eight new kinesin motor proteins. According to an exhaustive phylogenetic
comparison, Dictyostelium contains the same subset of kinesins that higher
eukaryotes need to perform mitosis. Some of the kinesins are implicated in
intracellular traffic and a small number have unpredictable functions.
Submitted by: Martin Kollmar [mako@nmr.mpibpc.mpg.de]
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The MADS-box transcription factor SrfA is required for actin cytoskeleton
organization and spore coat stability during Dictyostelium sporulation
Ricardo Escalante, Yohko Yamada, David Cotter, Leandro Sastre and Masazumi
Sameshima.
Mechanisms of Development, in press
The MADS-box transcription factor SrfA is involved in spore differentiation
in Dictyostelium (Escalante and Sastre, 1998). Mutant spores show an
altered morphology and loss of viability. A detailed structural analysis of
mutant spores has been performed to gain insight into the specific aspects
of spore differentiation in which SrfA is involved. Two main structural
defects have been observed. One is the formation of high order actin
structures, the so called actin rods. SrfA mutant spores showed the initial
stages of rod formation but no mature rods were found in older spores
either in the nucleus or the cytoplasm. Moreover, phosphorylation of actin,
that is believed to stabilize the actin rods, is strongly reduced in the
mutant. The other defect observed was the formation of the spore coat.
Young srfA-- spores show basically normal trilaminar coat structures
suggesting that release of prespore vesicles and basic assembly of the coat
takes place in the absence of SrfA. However, the outer layer gets wavier as
the spore ages and suffers a progressive degradation suggesting a late
defect in the stability of the spore coat. Taken together, these results
suggest that SrfA is involved in late events of spore maturation necessary
for spore stability.
Submitted by: Leandro Sastre [lsastre@iib.uam.es]
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[End Dicty News, volume 21, number 18]
