Mobilization of the non-conjugative plasmid RSF1010: A genetic analysis of its origin of transfer
โ Scribed by Derbyshire, Keith M. ;Willetts, Neil S.
- Publisher
- Springer
- Year
- 1987
- Tongue
- English
- Weight
- 836 KB
- Volume
- 206
- Category
- Article
- ISSN
- 0026-8925
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โฆ Synopsis
The oriT site of the broad host-range multicopy IncQ plasmid RSF1010 was cloned onto the 2.2 kb pBR322-derived vector pED825. By successive subcloning and construction of deletions, the oriT region was localised on an 80-88 bp segment of DNA. This segment was contained within the HaeII fragment of RSF1010 that is known to include the relaxation nick site. The oriT region was sequenced and inverted repeats and sequences homologous to the oriT regions of ColE1 and RK2 were identified. A striking 10 bp inverted repeat at one end of the 88 bp oriT segment may be important for recognition of oriT, and its possible role in transfer is discussed. As for other plasmids, the oriT region served as the site for recA-independent, transfer-dependent, site-specific recombination. This provides genetic evidence that strand breakage and re-joining occur at oriT during transfer. Mobilization was independent of transcription by RNA polymerase in the donor cell, as shown by the lack of effect of rifampicin. Inversion of the oriT site with respect to the plasmid oriV site showed that there was no functional dependence of oriT on oriV for synthesis of primers possibly involved in recipient conjugal DNA synthesis. Alternative mechanisms are discussed.
๐ SIMILAR VOLUMES
The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative. The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT). The DNA sequence of the mobilization region
The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion a