dictyNews Volume 27 Number 08

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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 Glšckner, 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] 
----------------------------------------------------------------------------- 

 
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]