Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon.
We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor). With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element. Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium. The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype. The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived. Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment. Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.[1]References
- Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon. Bohlander, F.A., Summers, A.O., Meagher, R.B. Gene (1981) [Pubmed]
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