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dictyNews Volume 28 Number 09
dictyNews
Electronic Edition
Volume 28, number 9
April 13, 2007
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 dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Ethylene induces zygote formation through an enhanced expression of zyg1 in
Dictyostelium mucoroides
Aiko Amagai, Sho-saku Soramoto, Shu-hei Saito and Yasuo Maeda
Department of Developmental Biology and Neurosciences, Graduate School of
Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan
Exp Cell Res., in press
We have previously demonstrated that a potent plant hormone, ethylene induces
sexual development including zygote formation in Dictyostelium cells, and
that a novel gene (zyg1) is also involved in zygote formation. Based on these
findings, the present work was mainly designed to reveal 1) the precise
relationship between the ethylene amount and zygote formation, and 2) the
relation of in situ ethylene synthesis to zyg1 expression, using transformants
that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene
biosynthesis. ACOOE cells overexpressing Dd-aco gene overproduced ethylene
and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells,
in which the expression of Dd-aco was suppressed by the RNAi method, showed
a reduced level of ethylene production, thus resulting in inhibition of
zygote formation. Importantly, the expression of zyg1 was affected by the
amount of ethylene produced: Zyg1 expression was augmented in ACOOE cells,
but was significantly suppressed in ACO-RNAi cells. In another experiment,
we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the
function of ethylene by binding specifically to ethylene receptors, greatly
suppresses zygote formation. These results indicate that ethylene is capable
of inducing zygote formation through the expression of zyg1.
Submitted by: Aiko Amagai [aiamagai@mail.tains.tohoku.ac.jp]
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A G protein coupled receptor with a lipid kinase domain
is involved in cell density sensing
Deenadayalan Bakthavatsalam1+, Derrick Brazill2, Richard H. Gomer3, Ludwig
Eichinger1, Francisco Rivero1* and Angelika A. Noegel1*
1 Center for Biochemistry, Institute for Biochemistry I, Medical Faculty and
Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
2 Center for the Study of Gene Structure and Function, Hunter College, City
University of New York, New York, USA
3 Howard Hughes Medical Institute and Department of Biochemistry and Cell
Biology, Rice University, Houston, Texas, USA
Current Biology, in press
One mechanism multicellular structures use for controling cell number [1, 2]
involves the secretion and sensing of a factor, like leptin [3] or myostatin
[4] in mammals. Dictyostelium cells secrete autocrine factors for sensing cell
density prior to aggregation and multicellular development [5, 6]. One of
these factors, CMF (conditioned medium factor), is a glycoprotein that enables
starving cells to respond to cAMP pulses [7, 8, 9]. Its actions are mediated
by two receptors. CMFR1 activates a G-protein independent signaling pathway
that regulates gene expression [10]. An unknown GÄ1-dependent receptor
activates phospholipase C (PLC), which, by an unknown mechanism, regulates
the lifetime of GÄ2-GTP [11, 12, 13]. Here we describe RpkA, an unusual
seven transmembrane receptor fused to a C-terminal PIP5 kinase domain that
localizes in membranes of a late endosomal compartment. Loss of RpkA resulted
in formation of persistent loose aggregates concomitant with altered expression
of cAMP-regulated genes. The developmental defect can be rescued by full length
RpkA and the transmembrane domain only. The PIP5 kinase domain, although
important for efficient targeting, is dispensable for the developmental role
of RpkA. rpkAÄ{ cells secrete and bind CMF but are unable to induce downstream
responses like production of inositol trisphosphate. Inactivation of Ga1, a
negative regulator of CMF signaling, rescued the developmental defect of the
rpkAÄ{ cells, suggesting that RpkA actions are mediated by this G protein.
RpkA constitutes an essential component of the cell density sensing mechanism
that regulates aggregation during Dictyostelium development.
Submitted by: Angelika A. Noegel [noegel@uni-koeln.de]
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Desalted deep sea water increases transformation and homologous recombination
efficiencies in Dictyostelium discoideum
Hidekazu Kuwayama and Akira Nagasaki
Journal of Molecular Microbiology and Biotechnology, in press
The life cycle of Dictyostelium discoideum consists of many cellular and
developmental aspects. By virtue of its relatively high transformation
efficiency and a small haploid genome, this organism has proven to be
advantageous for characterizing gene functions. However, a much higher
transformation efficiency is required as one of the prerequisites for
unravelling gene function on a genome-wide scale. In this study, we
describe the positive effect of desalted deep sea water, when used as a
solvent medium, on the transformation and homologous recombination
efficiencies in Dictyostelium. A standard Dictyostelium medium HL5
containing desalted deep sea water, HL5dsw, distinctly increased both the
transformation and homologous recombination efficiencies by approximately
2- to 3-fold. Furthermore, we observed that the growth of cells in HL5dsw
both before and after electroporation contributed to the increase in
transformation efficiency. These results indicate that a simple modification
of the solvent medium remarkably enhanced the isolation of transformants and
gene targeted clones, which had previously been difficult to isolate.
Submitted by: Hidekazu Kuwayama [hidekuwayama@biol.tsukuba.ac.jp]
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Evolution of size and pattern in the social amoebas
Review
Pauline Schaap
College of Life Sciences, University of Dundee, UK.
Bioessays, in press
A fundamental goal of biology is to understand how novel phenotypes evolved
through changes in existing genes. The dictyostelids or social amoebas
represent a simple form of multicellularity, where starving cells aggregate
to build fruiting structures. This review summarizes efforts to provide a
framework for investigating the genetic changes that generated novel
morphologies in the dictyostelids. The foundation is a recently constructed
molecular phylogeny of the dictyostelids, which was used to examine trends
in the evolution of novel forms and in the divergence of genes that shape
these forms. There is a major trend towards formation of large unbranched
fruiting bodies, which is correlated with the use of cyclic AMP (cAMP) as a
secreted signal to coordinate cell aggregation. The role of cAMP in
aggregation arose through co-option of a pathway that originally acted to
coordinate fruiting body formation. The genotypic changes that caused this
innovation and the role of dynamic cAMP signaling in defining fruiting body
size and pattern throughout Dictyostelid evolution are discussed.
Submitted by: Pauline Schaap [p.schaap@dundee.ac.uk]
--------------------------------------------------------------------------------
Protocols for growth and development of Dictyostelium discoideum
Petra Fey1, Anthony S. Kowal2, Pascale Gaudet1, Karen E. Pilcher1, and
Rex L. Chisholm1,2*
1dictyBase, Center for Genetic Medicine, Northwestern University
2 Center for Genetic Medicine, Northwestern University
Nat Protoc., in press
Dictyostelium discoideum, a unicellular organism capable of developing into a
multicellular structure, is a powerful model system to study a variety of
biological processes. Because it is inexpensive and relatively easy to grow,
Dictyostelium is also frequently used in teaching laboratories. Here we
describe conditions for successfully growing and developing Dictyostelium
cells and methods for long-term storage of Dictyostelium amoebae and spores.
Submitted by: Rex Chisholm [r-chisholm@northwestern.edu]
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Transformation of Dictyostelium discoideum with plasmid DNA
Pascale Gaudet, Karen E. Pilcher, Petra Fey, and Rex L. Chisholm*
dictyBase, 676 N. St. Clair St, Suite 1260, Chicago, IL, 60611
*corresponding author
Nat Protoc., in press
DNA-mediated transformation is one of the most widely used techniques to study
gene function. The eukaryote Dictyostelium discoideum is amenable to numerous
genetic manipulations that require insertion of foreign DNA into cells. Here we
describe two commonly used methods to transform Dictyostelium cells: calcium
phosphate precipitation, resulting in high copy number transformants, and
electroporation, an effective technique for producing single integration
events into genomic DNA. Single integrations are required for gene disruption
by homologous recombination. We also discuss how different selection markers
affect vector copy number in transformants and explain why blasticidin has
become the preferred selectable marker for making gene knock-outs. Both
procedures can be accomplished in less than two hours of hands-on time;
however, the calcium-phosphate precipitation method contains several
incubations, including one of at least four hours, so the total time required
for the transformation is approximately 8 hours.
Submitted by: Rex Chisholm [r-chisholm@northwestern.edu]
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A reliable general purpose method for extracting genomic DNA from
Dictyostelium cells.
Karen E. Pilcher1, Petra Fey1, Pascale Gaudet1, Anthony S. Kowal2, and
Rex L. Chisholm1,2*
*corresponding author
1dictyBase, Center for Genetic Medicine, Northwestern University
2 Center for Genetic Medicine, Northwestern University
*corresponding author
Nat Protoc., in press
In this protocol, we present a standard method for extracting DNA from cells
of the social amoeba Dictyostelium discoideum. While this procedure is similar
to other phenol:chloroform-based purification methods, it is modified to
account for the high level of carbohydrate and nucleases found in
Dictyostelium cells. Genomic DNA can be isolated from wild type and
genetically modified cells using the described protocol, allowing molecular
genetic analyses to be performed. Following cell lysis, nucleic acid
extraction, and precipitation, the isolated DNA is suitable for digestion by
restriction enzymes, amplification by PCR, and Southern blotting. This
procedure takes approximately 3 hours to complete.
Submitted by: Rex Chisholm [r-chisholm@northwestern.edu]
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A general purpose method for extracting RNA from Dictyostelium cells
Karen E. Pilcher, Petra Fey, Pascale Gaudet, and Rex L. Chisholm*
dictyBase, 676 N. St. Clair St, Suite 1260, Chicago, IL, 60611
*corresponding author
Nat Protoc., in press
Here we present a protocol for the extraction of RNA from Dictyostelium
discoideum. Dictyostelium is a social amoeba which undergoes a basic
developmental program, and therefore analysis of RNA levels over a time course
is a commonly used technique. This procedure is similar to other guanidine
thiocyanate-based methods; however, it has been adjusted because of the large
quantities of carbohydrate and nucleases found in Dictyostelium cells. After
cell lysis and phenol:chloroform extraction, the resulting high quality RNA
isolated with the described protocol allows the molecular genetic analysis of
wild type and genetically modified cells. The purified RNA can be used for
analyses such as Northern blotting, RT-PCR, and microarrays. From start to
finish, this procedure requires approximately two hours to complete.
Submitted by: Rex Chisholm [r-chisholm@northwestern.edu]
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[End dictyNews, volume 28, number 9]
