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Precise Excision (precise + excision)
Selected AbstractsNew insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaeaMOLECULAR MICROBIOLOGY, Issue 1 2005Zachary D. Blount Summary Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II ,Miniature Inverted-repeat Transposable Element' (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes. [source] Regulated site-specific recombination of the she pathogenicity island of Shigella flexneriMOLECULAR MICROBIOLOGY, Issue 5 2004Harry Sakellaris Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3, terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase-mediated excision occurs via recombination between a 22 bp sequence at the 3, terminus of pheV and an imperfect direct repeat at the pheV -distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase-mediated integration into the chromosome. The circle junction consists of the pheV -proximal and pheV -distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3, terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase-dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2. [source] A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants,PLANT BIOTECHNOLOGY JOURNAL, Issue 8 2008Vesna Djukanovic Summary The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-µm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system. [source] | ||||||||||