Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid
- PMID: 342513
- PMCID: PMC222101
- DOI: 10.1128/jb.133.2.884-896.1978
Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid
Abstract
Bacteriophage lambdab2att2 [lambdab2cI857intam6(deltabioAB)bioFCD+uvrB+phr+] codes for a function(s) that confers UV resistance (Uvr+) and reactivation of irradiated phage (Hcr+) to an Uvr-Hcr-Escherichia coli strain. It was demonstrated that these functions are expressed under the control of bacterial regulatory elements located on lambdab2att2 DNA. The location of the E. coli uvrB gene on the DNA of this transducing phage was established by heteroduplex and restriction-enzyme analyses. Recombinant DNA molecules were constructed in vitro from plasmid pMB9 (Tcr), as the vector, and an EcoRI fragment (Eco-RI-F) of lambdab2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 X 10(6) and replicates in a relaxed fashion. Transformation of E. coli uvrB with plasmid pNP5 resulted in clones that are Uvr+ Tcr. Irradiation of bacteria transformed with plasmid pNP5 with low UV doses revealed a complete restoratation of the Uvr+ phenotype by the presence of the cloned EcoRI-F DNA, while only a partial restoration was observed after irradiation with high UV doses. Likewise, the Hcr+ character was also partially restored due to the presence of pNP5. No correlation was found between the acquired Uvr+, Hcr+ properties, and the presence of correndonuclease II activity in an extract of bacteria that harbor plasmid pNP5.
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