CSM News Electronic Edition Volume 7, number 6 Sept, 7, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Replicon Rescue - A novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA. Tara L. McMahon, Zofia Wilczynska, Christian Barth, Derek J. Fraser, Luisa Pontes and Paul R. Fisher* School of Microbiology , La Trobe University, Bundoora, Melbourne, Australia Nucleic Acids Research, in press. Abstract A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two-fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal ori from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis deficient mutant of Dictyostelium. ------------------------------------------------------------------ Efficient Circularization in E. coli of Linear Plasmid Multimers from Dictyostelium discoideum genomic DNA C. BARTH, Z. WILCZYNSKA, L. PONTES, D.J. FRASER AND P.R. FISHER School of Microbiology, La Trobe University, Bundoora 3083, Melbourne, Australia Plasmid, in press. ABSTRACT Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of Dictyostelium discoideum. --------------------------------------------------------------------- [End CSM News, volume 7, number 6]