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. ============= Abstracts ============= 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] ----------------------------------------------------------------------------- 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] ============================================================================== [End dictyNews, volume 27, number 8]