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Transposable elements (i.e. insertion sequences and transposons) are components of nearly all bacterial genomes. The majority of these elements have been identified as a result of various sequencing projects. However, in most cases, their activity was not experimentally confirmed. For this reason several strategies have been developed that allow direct cloning and identification of functional transposable elements. Most of the methods are based on the ability of transposable elements to inactivate or activate particular genes by insertion. In this review we describe and critically discuss different cloning strategies that employ various entrapment vectors, carrying (i) conditionally lethal genes, (ii) antibiotic selection cartridges, (iii) promoter-less genes or (iv) suicide replicons. These tools, besides facilitating the identification of new transposable elements, also enable the investigation of various DNA rearrangement mutations, which are related to the transposition process.
The replication system of plasmid pMTH4 (22 kb) of dichloromethane-degrading Paracoccus methylutens DM12 (Alphaproteobacteria) has been cloned within a mini-replicon pMTH100 (4.7 kb) and preliminarily characterized. Functional analysis, performed with a series of mutated plasmid mini-derivatives, showed that the replicator region consists of three elements: (i) gene repA coding for a replication initiation protein RepA, (ii) origin of replication (oriV), placed in the promoter region of repA and containing a set of imperfect directly repeated sequences (iterons) together with putative DnaA and IHF-binding DNA sequences as well as (iii) an enhancer (0.65 kb) upstream of oriV. We showed that the enhancer was not crucial for plasmid replication, however, it was necessary to assure the proper plasmid copy number. Additionally its presence has increased the strength of a determinant of incompatibility (located within the oriV region) as well as the level of transcription carried from the repA promoter. The enhancer region was shown not to encode any proteins or promoter sequences. We speculate that this region might constitute a site of binding of plasmid or host-encoded proteins that are able to interact with the origin, which positively regulates the initiation of replication.
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