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dictyNews Volume 20 Number 05
Dicty News
Electronic Edition
Volume 20, number 5
March 22, 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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** 10th ANNIVERSARY EDITION **
** **
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This edition of the Dicty News represents the 10th year of regular
publication of the Electronic Edition. The first issue was March
15, 1993.
Thanks to everyone for their contributions and support!
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Abstracts
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Macroautophagy is Required for Multicellular Development of the Social
Amoeba Dictyostelium discoideum
Grant P. Otto1, Mary Y. Wu1, Nevzat Kazgan1, O. Roger Anderson2, and
Richard H. Kessin1
1) Department of Anatomy and Cell Biology, Columbia University 630 West
168th St., New York, NY, 10032, USA
2) Department of Biology, Lamont-Doherty Earth Observatory Columbia
University, Palisades, NY, 10964, USA
J. Biol. Chem., in press
Macroautophagy is a mechanism employed by eukaryotic cells to recycle
non-essential cellular components during starvation, differentiation,
and development. Two conjugation reactions related to ubiquitination
are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p
conjugation to the lipid phosphatidylethanolamine. These reactions require
the action of the E1-like enzyme Apg7p, and the E2-like enzymes, Apg3p and
Apg10p. In Dictyostelium, development is induced by starvation, conditions
under which autophagy is required for survival in yeast and plants. We
have identified Dictyostelium homologues of 10 budding yeast autophagy
genes. We have generated mutations in apg5 and apg7 that produce defects
typically associated with an abrogation of autophagy. Mutants are not
grossly affected in growth, but survival during nitrogen starvation is
severely reduced. Starved mutant cells show little turnover of cellular
constituents by electron microscopy, whereas wild-type cells show
significant cytoplasmic degradation and reduced organelle number. Bulk
protein degradation during starvation-induced development is reduced in
the autophagy mutants. Development is aberrant; the autophagy mutants do
not aggregate in plaques on bacterial lawns, but they do proceed further
in development on nitrocellulose filters, forming defective fruiting
bodies. The autophagy mutations are cell autonomous, since wild-type
cells in a chimaera do not rescue development of the autophagy mutants.
We have complemented the mutant phenotypes by expression of the cognate
gene fused to GFP. A GFP fusion of t he autophagosome marker Apg8
mislocalizes in the two autophagy mutants. We show that the Apg5-Apg12
conjugation system is conserved in Dictyostelium.
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Cultivation of Dictyostelium discoideum in immobilized form by colonization
of porous supports
Usama Beshay, Karl Friehs, Abd-El-Mouty Azzam and Erwin Flaschel:
Bielefeld University, Faculty of Technology, D-33594 Bielefeld, Germany
Process Biochemistry (in press)
Abstract
Dictyostelium discoideum is a social amoeba. It shows promising
characteristics as an expression system for the production of recombinant
glycoproteins owing to its ability to perform a whole spectrum of post-
translational modifications. However, its application is seriously
affected by slow growth rates as well as low maximal cell densities.
Since the technique of immobilization often is applied in order to achieve
high cell concentrations, D. discoideum has been cultivated in the
presence of different porous supports with the aim of studying its growth
in immobilized state. On small scale a shake flask cultivation system
with external loop was applied for this purpose. On larger scale a
bioreactor was coupled with an external loop of a column containing a
fixed bed of the porous support. During growth on standard axenic
(soluble) media cell densities in the pores of the supports were attained,
which were more than one magnitude higher than those normally obtained
during suspension culture. Electron micrograph images gave an idea, how
D. discoideum may manage to colonize porous support particles
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Sequence and Structure of the Extrachromosomal Palindrome Encoding the
Ribosomal RNA Genes in Dictyostelium
Richard Sucgang, Guokai Chen, Wen Liu, Ryan Lindsay, Jing Lu, Donna Muzny,
Gad Shaulsky, William Loomis, Richard Gibbs and Adam Kuspa
Nucleic Acids Research, in press
Ribosomal RNAs are encoded by multicopy families of identical genes. In
Dictyostelium and other protists, the rDNA is carried on extrachromosomal
palindromic elements that comprise up to 20% of the nuclear DNA. We present
the sequence of the 88-kb Dictyostelium rDNA element, noting that the rRNA
genes are likely to be the only transcribed regions. By interrogating a
library of ordered YAC clones, we provide evidence for a chromosomal copy
of the rDNA on chromosome 4. This locus may provide master copies for the
stable transmission of the extrachromosomal elements. The extrachromosomal
elements were also found to form chromosome-sized clusters of DNA within
nuclei of nocodazole-treated cells arrested in mitosis. These clusters
resemble true chromosomes and may allow the efficient segregation of the
rDNA during mitosis. These rDNA clusters may also explain the cytological
observations of a seventh chromosome in this organism.
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A novel gene trap method using terminator-REMI and 3 rapid amplification
of cDNA ends (RACE) in Dictyostelium
Kosuke Takeda a, 1, Tamao Saito a, Tomoyuki Tanaka a, Takahiro Morio b,
Mineko Maeda c, Yoshimasa Tanaka b and Hiroshi Ochiai a, *
a) Division of Biological Sciences, Graduate School of Science, Hokkaido
University. Sapporo, Hokkaido 060-0810, Japan
b) Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan
c) Division of Biological Sciences, Graduate School of Science, Osaka
University, Japan
Gene, in press
Abstract
We describe a novel restriction enzyme-mediated integration (REMI) method
for gene trapping in Dictyostelium based on the use of a terminator-
deficient vector. The vector has a blasticidin deaminase (bsr) gene as a
selectable marker but lacks a terminator containing a poly(A) addition
signal (AATAAA). Thus, the vector was expected to integrate into the
coding region of a gene to create a fusion transcript flanked by the 3
proximal region of the trapped gene. The trapped gene can be identified
by simply amplifying the fusion transcript by 3 rapid amplification of
cDNA ends (3 -RACE). In the analysis of 35 integration events into known
genes, the vectors were found to be integrated 20 times in close proximity
to the 3 ends of the genes and in the direction of transcription. This
strictly localized insertion seemed to be mediated by negative selection
via the surveillance system referred to nonsense-mediated mRNA decay. In
contrast, in 15 events the vector integrated in the opposite direction to
transcription and at random positions throughout the coding sequence.
Analysis of the trapped 3 sequences showed that the transcription of the
fusion gene terminated prematurely without the apparent use of an
endogenous terminator; nevertheless the transcript did exhibit a poly(A)
tail. Based on these results, we designated the method terminator-REMI.
Using this method, we have generated a library of tagged Dictyostelium
clones from which we have thus far isolated 242 developmental mutants.
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A Novel Developmental Mechanism in Dictyostelium Revealed in a Screen for
Communication Mutants
Kirsten Kibler (1,2), Tu-Lan Nguyen (2,3), Jessica Svetz (2), Nancy Van
Driessche (1,2), Miroslava Ibarra (2), Christopher Thompson (2), Chad Shaw
(2), and Gad Shaulsky (1,2,4)
1 Graduate Program in Developmental Biology
2 Department of Molecular and Human Genetics
3 Summer Medical And Research Training (SMART) Program
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
4 Corresponding Author. email: gadi@bcm.tmc.edu; phone: 713 798 8082;
FAX 713 798 1021.
Developmental Biology, in press
Abstract
We performed a screen for signaling genes by selecting mutant strains of
Dictyostelium that fail to develop spores in a pure population but
sporulate well in chimerae with wild type cells. We found 9 strains whose
sporulation was induced up to ten million fold in chimerae. Most strains
were also able to sporulate in chimerae with each other, but two pairs
failed to do so, suggesting that the genes in each pair participate in
the production of one signal. One of the pairs, comD and comB, is
described in detail. Sequence analysis revealed that both genes encode
putative membrane proteins. ComD is predicted to have 15 transmembrane
domains and ComB has a region of high similarity to the Rab family of
small GTPases and one transmembrane domain. Similarities between the
developmental regulation and cell-type specificity of the genes'
expression, the terminal developmental morphology and the expression
pattern of cell-type specific markers in the mutants suggest that comD
and comB participate in one signal production pathway. This idea is also
supported by a high similarity between the global transcriptional
profiles of the mutant strains. Differences between the mutant phenotypes
late in development suggest that comD and comB participate in separate
processes as well. comD has a cell-autonomous role in the specialization
of a novel of a prespore cell type whereas comB has a cell-autonomous
role in prestalk A cell differentiation.
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[End Dicty News, volume 20, number 5]
