Mapping of theben, antandcatgenes ofPseudomonas aeruginosaand evolutionary relationship of thebenregion ofP. aeruginosaandP. putida

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

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Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome

Gralton

Campbell

Neidle

1997

Microbiology

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The natural transformability of the soil bacterium Acinetobacter sp. ADPl (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 378021 91 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease Notl. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered Not1 cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of Notl, Ascl and I-Ceul recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColEl-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADPI.

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Mapping of theben, antandcatgenes ofPseudomonas aeruginosaand evolutionary relationship of thebenregion ofP. aeruginosaandP. putida

Cited by 9 publications

References 21 publications

Genomic Insights in the Metabolism of Aromatic Compounds in Pseudomonas

Genomic Insights in the Metabolism of Aromatic Compounds in Pseudomonas

Transposon-mediated generation of targeting vectors for the production of gene knockouts

Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome

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