Copy Link
Add to Bookmark
Report
dictyNews Volume 21 Number 13
Dicty News
Electronic Edition
Volume 21, number 13
October 17, 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.
=============
Abstracts
=============
Identification of Dictyostelium discoideum
developmentally regulated genes whose expression is dependent on the
MADS-box transcription factor SrfA
Ricardo Escalante, Nicolas Moreno, and Leandro Sastre
Eukaryotic Cell, in press
The MADS-box transcription factor SrfA is required for spore
differentiation in Dictyostelium discoideum. srfA null strains form rounded
spores that do not resist adverse environmental conditions. Five genes
whose expression is dependent on SrfA have been isolated by differential
hybridization. One of these genes, sigC, is identical to phg1b, previously
characterized in mutants with altered adhesive properties and found to
encode a nine transmembrane domain protein. This gene is transcribed into
two mRNAs, as the result of alternative splicing of two internal exons. The
slower migrating mRNA codes for a shorter protein that lacks the first
transmembrane fragment and is not expressed in srfA null strains. The other
four genes (sigA, B, D and 45D) are expressed only during late
developmental stages. In situ hybridization experiments showed that
expression of sigA, B and D is restricted to the sorus of developing
structures. sigA codes for a homolog of malate dehydrogenase that converts
pyruvate to malate to replenish the tricarboxylic acid cycle. sigB encodes
a protein with significant similarity to the GP63 metalloproteinase of
Leishmania, leishmanolysin. The sequence of sigD is highly similar to that
of several spore coat proteins of D. discoideum and it may play a role in
that structure. The gene 45D codes for a RNA-binding protein homologue
whose expression is also dependent on the GATA transcription factor stalky
(StkA). The expression of sigB is also dependent on both SrfA and StkA. The
expression of 45D, but not of sigA, B, C and D, can be induced in srfA null
cells by constitutive PKA activation. Strains in which either sigA, sigB or
sigD are disrupted were isolated and found to form spores that are not
detectably different from those of wild type strains.
Submitted by: Leandro Sastre [lsastre@iib.uam.es]
-----------------------------------------------------------------------------
Regulatory Mechanism of Dictyostelium Myosin Light Chain Kinase A
Hiroshi Tokumitsu*à, Naoya Hatano¤, Hiroyuki Inuzuka*, Yumi Ishikawa*,
Taro Q. P. Uyeda¦, Janet L. Smith , and Ryoji Kobayashi*
*Department of Signal Transduction Sciences and ¤Department of Cell
Physiology, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa
761-0793, Japan, ¦Gene Discovery Research Center, National Institute of
Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562,
Japan, Boston Biomedical Research Institute, 64 Grove St. Watertown,
MA 02472-2829, U.S.A
J. Biol. Chem., in press
In this report, we have examined the activation mechanism of Dictyostelium
MLCK-A by using constitutively active Ca2+/calmodulin-dependent protein
kinase kinase (CaM-KKc) as a surrogate MLCK-A kinase. MLCK-A was
phosphorylated at Thr166 by CaM-KKc, resulting in a ~140-fold increase in
catalytic activity, using intact Dictyostelium myosin II. Recombinant
Dictyostelium MRLC and Kemptamide were also readily phosphorylated by the
activated MLCK-A. Mass spectrometry analysis revealed that
E. coli-expressed MLCK-A was autophosphorylated at Thr289 and subsequent to
Thr166 phosphorylation, MLCK-A also undergoes a slow rate of
autophosphorylation at multiple Ser residues. Using site-directed
mutagenesis, we showed that autophosphorylation at Thr289 is required for
efficient phosphorylation and activation by an upstream kinase. By
performing enzyme kinetics on a series of truncated MLCK-As, we found that
residues 283-288 function as an autoinhibitory domain, and that autoinhibition
is fully relieved by Thr166 phosphorylation. Simple removal of this region
results in a significant increase in the kcat of MLCK-A, however, it does not
generate maximal enzymatic activity. Together with the results of our kinetic
analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation
of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289
results in generation of maximum MLCK-A activity through both release of an
autoinhibitory domain from its catalytic core and a further increase
(15-19-fold) in the kcat of the enzyme.
Submitted by: Janet Smith [smith@bbri.org]
-----------------------------------------------------------------------------
Evolutionary questions raised by cellular slime mould development
Sonia Kaushik(*) and Vidyanand Nanjundiah
Indian Institute of Science, Bangalore 560012, India
(*) Current address: Department of Microbiology, La Trobe University, VIC
3086, AUSTRALIA.
Proceedings of the Indian National Science Academy, in press
REVIEW
The cellular slime moulds (CSMs) are amoeboid organisms whose life cycle can
be viewed in two ways. Firstly, because free-living amoebae come together to
build bodies, they are ideal models for studying multicellular development
in terms of the properties of single cells. Secondly, coming together and
participating in an integrated unit implies social behaviour. Consequently
differentiation (especially in the advanced CSMs) can be seen as a form of
division of labour in which only some amoebae get to transmit their genes to
the next generation. Viewed thus, their life cycle is ideally suited for
studying the evolutionary basis of cooperation with some members of the
cooperating group exhibiting altruistic behaviour. The present review takes
the second approach. We examine alternative explanations for social
behaviour (i.e., multicellular development) based on individual-level and
group (including kin-group) selection. Non-clonal fruiting bodies are likely
to be common in nature; we show a case with at least nine genotypes. The
CSMs display both individual and group-level adaptations and both levels of
selection operate in their appropriate contexts. The review ends with
questions for the future and indicates how studies of CSM development might
help to explain the evolution of altruism in this group.
Submitted by: Vidyanand Nanjundiah [vidya@ces.iisc.ernet.in]
-----------------------------------------------------------------------------
Calcium regulates the expression of a Dictyostelium discoideum asparaginyl
tRNA synthetase gene.
Jyoti K. Jaiswal* and Vidyanand Nanjundiah
Indian Institute of Science, Bangalore 560012, India.
* (Current address) The Rockefeller University, 1230 York Avenue, Box 304,
New York, NY 10021, USA
Journal of Biosciences, in press
In a screen for calcium-regulated gene expression during growth and
development of Dictyostelium discoideum we have identified an asparaginyl
tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified
in this organism. The gene shows many unique features. It is repressed by
lowering cellular calcium, making it the first known calcium-regulated tRNA
synthetase. Also, despite the calcium-dependence, its expression is
unaltered during the cell cycle, making it the first D. discoideum gene to
show a calcium-dependent but cell cycle phase-independent expression.
Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher
sequence similarity to Gln tRNA synthetases than to other Asn tRNA
synthetases. These unique features of the AsnRS from this primitive
eukaryote point to a novel mechanism that regulates the components of the
translation machinery and gene expression by calcium, and also indicate a
link between the evolution of GlnRS and AsnRS in eukaryotes.
Submitted by: Vidyanand Nanjundiah [vidya@ces.iisc.ernet.in]
-----------------------------------------------------------------------------
A bZIP/bRLZ Transcription Factor Required for DIF Signaling in Dictyostelium
Christopher R.L. Thompson (1), Qing Fu (1), Caroline Buhay (1), Robert R.
Kay (2) and Gad Shaulsky(1)
1 Department of Molecular and Human Genetics,
Baylor College of Medicine, One Baylor Plaza,
Houston, Texas, USA 77030
2 MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH UK
Development, in press
The intermingled differentiation and sorting out of Dictyostelium prestalk-O
and prespore cells requires the diffusible signaling molecule DIF-1 and
provides an example of a spatial information-independent patterning mechanism.
To further understand this patterning process, we used a genetic selection to
isolate mutants in the DIF-1 response pathway. The disrupted gene in one
such mutant, dimA-, encodes a bZIP/bRLZ transcription factor, which is
required for every DIF-1 response investigated. Furthermore, the dimA- mutant
shows strikingly similar developmental defects to the dmtA- mutant, which is
specifically defective in DIF-1 synthesis. However, key differences exist:
(1) the dmtA- mutant responds to DIF-1 but does not produce DIF-1 (2) the
dimA- mutant produces DIF-1 but does not respond to DIF-1 (3) the dimA- mutant
exhibits cell autonomous defects in cell type differentiation. These results
suggest that dimA encodes the key transcriptional regulator required to
integrate DIF-1 signaling and subsequent patterning in Dictyostelium.
Submitted by: Gadi Shaulsky [gadi@bcm.tmc.edu]
-----------------------------------------------------------------------------
Two Phases of Actin Polymerization Display Different Dependences on
PI(3,4,5)P3 Accumulation and Have Unique Roles during Chemotaxis
Lingfeng Chen, Chris Janetopoulos, Yi Elaine Huang, Miho Iijima, Jane
Borleis and Peter N. Devreotes
Department of Cell Biology, Johns Hopkins University, School of Medicine
Baltimore, MD, 21205
MBC, in press
The directional movement of cells in chemoattractant
gradients requires sophisticated control of the actin cytoskeleton. Uniform
exposure of D. discoideum amoebae as well as mammalian leukocytes to
chemoattractant triggers two phases of actin polymerization. In the initial
rapid phase, motility stops and the cell rounds up. During the second slow
phase, pseudopodia are extended from local regions of the cell perimeter.
These responses are highly correlated with temporal and spatial
accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of
specific PH domains to the membrane. The slower phase of PI accumulation
and actin polymerization is more prominent in less differentiated,
unpolarized cells, is selectively increased by disruption of PTEN, and is
relatively more sensitive to perturbations of PI3K. Optimal levels of the
second responses allow the cell to respond rapidly to switches in gradient
direction by extending lateral pseudopods. Consequently, PI3K inhibitors
impair chemotaxis in wild type cells but partially restore polarity and
chemotactic response in pten- cells. Surprisingly, the fast phase of
PI(3,4,5)P3 accumulation and actin polymerization, which is relatively
resistant to PI3K inhibition, can support inefficient but reasonably
accurate chemotaxis.
Submitted by: Lingfeng Chen [lche1@jhmi.edu]
-----------------------------------------------------------------------------
Hypertonic signal promotes stability of Dictyostelium spores via a PKA
independent pathway
Yohko Yamada and Masazumi Sameshima
The Tokyo Metropolitan Institute of Medical Science, Electron Microscopy
Center
FEMS Microbiol. Lett., in press
Differentiation of Dictyostelium spores is initiated with rapid
encapsulation of prespore cells under the control of cAMP dependent protein
kinase (PKA), followed by further maturation process that involves
cytoskeletal organization. Constitutive activation of PKA induces precocious
formation of viable spores in development and confer cells the ability to
encapsulate under specific submerged conditions. In this study we show that
the stability of these spores depended on high osmotic condition during spore differentiation, indicating that hypertonic signal is required in addition to
PKA to induce matured stable spores. Formation of stable spores under a high
osmotic condition required high cell density, suggesting the involvement of
additional cellular signaling.
Submitted by: Yohko Yamada [yyamada@rinshoken.or.jp]
-----------------------------------------------------------------------------
A/T-rich inverted DNA repeats are destabilized by chaotrope-containing buffer
during purification using silica gel membrane technology.
M. Prevorovsky and F. Puta
BioTechniques 35:698-702 (October 2003) Ê
The recovery of electrophoretically separated DNA fragment(s) from agarose
gel is afrequently used method in most gene engineering procedures. These
techniques wereconsiderably simplified by the implementation of commercial
kits based on silicamicroparticles or glass fibers. In this report we show
that during extraction the chaotropecontainingagarose-melting buffer can
cause destabilization of short A/T rich DNA fragmentsof inverted
repeat-structure. We propose that destabilized inverted repeats formed
hairpinsafter the alleviation of denaturing conditions. Expression of RNA
molecules of invertedrepeat-structure is the method of permanent gene
silencing by RNA interference (RNAi) invarious organisms, so we believe
that our findings are of interest to researchers conductingRNAi experiments
and/or working with, e.g., Plasmodium and other unicellular pathogens,
characterized by A/T rich genomes.
Submitted by: Frantisek Puta [puta@natur.cuni.cz]
===============================================================================
[End Dicty News, volume 21, number 13]
