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dictyNews Volume 27 Number 08
dictyNews
Electronic Edition
Volume 27, number 8
September 8, 2006
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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Optimization of a large-scale gene disruption protocol in Dictyostelium
and analysis of conserved genes of unknown function
Patricia Torija, Alicia Robles and Ricardo Escalante
BMC Microbiology 2006, 6:75, in press
(available athttp://www.biomedcentral.com/bmcmicrobiol/)
Background: Development of the post-genomic age in Dictyostelium will
require the existence of rapid and reliable methods to disrupt genes
that would allow the analysis of entire gene families and perhaps the
possibility to undertake the complete knock-out analysis of all the
protein-coding genes present in Dictyostelium genome.
Results: Here we present an optimized protocol based on the previously
described construction of gene disruption vectors by in vitro
transposition. Our method allows a rapid selection of the construct by a
simple PCR approach and subsequent sequencing. Disruption constructs
were amplified by PCR and the products were directly transformed in
Dictyostelium cells. The selection of homologous recombination events
was also performed by PCR. We have constructed 41 disruption vectors to
target genes of unknown function, highly conserved between Dictyostelium
and human, but absent from the genomes of S. cerevisiae and S. pombe. 28
genes were successfully disrupted.
Conclusions: This is the first step towards the understanding of the
function of these conserved genes and exemplifies the easiness to
undertake large-scale disruption analysis in Dictyostelium.
Submitted by: Ricardo Escalante [rescalante@iib.uam.es]
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On the role of RNA polymerase III transcription factors in the selection of
integration sites by the Dictyostelium non-LTR retrotransposon TRE5-A
Oliver Siol, Moustapha Boutliliss, Thanh Chung, Gernot Glckner, Theodor
Dingermann and Thomas Winckler
Mol. Cell. Biol., in press
In the compact Dictyostelium discoideum genome, non-long terminal repeat
(non-LTR) retrotransposons known as TREs avoid accidental
integration-mediated gene disruption by targeting the vicinity of tRNA
genes. In this study we provide the first evidence that proteins of a
non-LTR retrotransposon interact with a target-specific transcription factor
to direct its integration. We applied an in vivo selection system that
allows for the isolation of natural TRE5-A integrations into a known genomic
location upstream of tRNA genes. TRE5-A frequently modified the integration
site in a way characteristic of other non-LTR retrotransposons by adding
non-templated extra nucleotides and generating small and extended target
site deletions. Mutations within the B box promoter of the targeted tRNA
genes interfered with both the in vitro binding of RNA polymerase III
transcription factor TFIIIC and the ability of TRE5-A to target these genes.
An isolated B box was sufficient to enhance TRE5-A integration in the
absence of a surrounding tRNA gene. The pol III-transcribed ribosomal 5S
gene recruits TFIIIC in a B box-independent manner, yet it was readily
targeted by TRE5-A in our assay. These results suggest a direct role of an
RNA polymerase III transcription factor in the targeting process.
Submitted by: Thomas Winckler [t.winckler@uni-jena.de]
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A Putative Ariadne-Like Ubiquitin Ligase Is Required for Dictyostelium
discoideum Development
Nathaniel Whitney,1 Lacey J. Pearson,1 Ryan Lunsford,1 Lisa McGill,1
Richard H. Gomer,2 and David F. Lindsey1
Department of Biological Sciences, Walla Walla College, College Place,
Washington 99324,1 and Howard Hughes Medical Institute, Department of
Biochemistry and Cell Biology, Rice University, Houston, Texas 770052
The Dictyostelium rbrA gene encodes a putative Ariadne ubiquitin ligase.
rbrA- cells form defective slugs that cannot phototax. Prestalk cell
numbers are reduced in rbrA- slugs, and these prestalk cells do not
localize to the tip of slugs. Chimeric slugs containing wild-type cells
could phototax and form fruiting bodies.
Submitted by: David Lindsey [lindda@wwc.edu]
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[End dictyNews, volume 27, number 8] 
