dictyNews Electronic Edition Volume 37, number 12 November 11, 2011 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. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Highly effective removal of floxed Blasticidin S resistance cassettes from Dictyostelium discoideum mutants by extrachromosomal expression of Cre Joern Linkner, Benjamin Nordholz, Alexander Junemann, Moritz Winterhoff and Jan Faix Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany EJCB, in press The inactivation of proteins in cells is inevitable to study their physiological role in various cellular processes. In contrast to strategies to alter the amount of active proteins in cells, only a gene knockout guarantees complete removal of the protein of interest. For Dictyostelium discoideum cells, the gene replacement construct typically consists of a Blasticidin S resistance (Bsr) cassette flanked by fragments of the target gene to allow insertion by homologous recombination. More advanced knockout constructs additionally carry loxP sites on both sides of the Bsr cassettes for subsequent removal of the selection marker by transient expression of Cre recombinase, thus allowing generation of multiple knockouts using just a single selection marker. However, due to its design, the available neomycin selection-based Cre expression plasmid occasionally tends to integrate into the genome and also yield only a moderate number of transfectants in liquid media. In some cases, for instance in SCAR-null cells, it was not possible to remove the Bsr cassette without stable integration of the Cre expression vector into the genome. To circumvent these difficulties we designed the extrachromosomal Cre-recombinase expression vector pTX-NLS-Cre. We verified the greatly improved efficacy of this novel Cre-loxP approach by removal of the Bsr cassette in five different cell lines including the SCAR-null mutant. By consequence, this vector will be a highly valuable means for the rapid generation of single or multiple mutants remaining sensitive to the most reliable selection markers Blasticidin S and neomycin. Submitted by Jan Faix [aix.jan@mh-hannover.de] -------------------------------------------------------------------------------------- Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism Anne Krueger, Petros Batsios, Otto Baumann, Eva Luckert, Heinz Schwarz, Reimer Stick, Irene Meyer*, Ralph Graef* Dept. of Cell Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14469 Potsdam-Golm, Germany Mol. Biol., Cell in press Lamins build the nuclear lamina and are required for chromatin organization, gene expression, cell cycle progression and mechanical stabilization. Despite these universal functions, lamins have so far been found only in metazoans. We have identified a protein NE81 in Dictyostelium, whose properties justify denomination as a lamin-like protein in a lower eukaryote. This is based on its primary structure, subcellular localization, regulation during mitosis, and the requirement of the C-terminal CaaX box as a post-translational processing signal for proper localization of the protein. Our knock-out and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization and mechanical stability of cells. All our results are in agreement with a role of NE81 in formation of a nuclear lamina. This function is corroborated by localization of Dictyostelium NE81 at the nuclear envelope in human cells. The discovery of a lamin-like protein in a unicellular organism is not only intriguing in the light of evolution, it may also provide a simple experimental platform for studies of the molecular basis of laminopathies. Submitted by Ralph Graef [rgraef@uni-potsdam.de] ============================================================== [End dictyNews, volume 37, number 12]