Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid

Abstract

Bacteriophage Ab2ate [Ab2cI857intam6(AbioAB)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 Xb2att2 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 (EcoRI-F) of Xb2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 x 106 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 restoration 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. This work HP3404 KMBL1001(pNP5 Tcr)Uvr+ This work a All bacterial strains are F- .

Relief of polarity caused by transposon Tn5: application in mapping a cloned region of the Escherichia coli uvrB locus essential for UV resistance

“…We have argued before that this segr6ent is not related to the uvrB locus. Hence, this result confirms our previous conclusion on the composition of the cloned EcoRI fragment (Pannekoek et al, 1978). Transformants which displayed a AprTcrcolimmuv s phenotype were further examined.…”

“…Plasmid pNP5 has been constructed by introduction of an EcoRI fragment (2.5 kb), derived from a uvrB.transducing phage Xb 2att 2 (~ 2intam6( ~bioAB ) bioFCD÷uvrB÷cI857), into the unique EcoRI site of plasmid pMB9 (5.3 kb), which carries genes specifying Tc r and colicin immunity (col lmm) (Rodriguez et al, 1976;Pannekoek et al, 1978). The cloned EcoRI fragment contains, besides the uvrB locus, a segment of DNA, located between 68.3% and 75.4% of the pNP5 map, which originates from the opposite side of the X attachment site on the chromosome as the uvrB 53 locus ( Fig.…”

“…Plasmid pNP5 lacks the uvrB promoter, but contains the genetic information to fully complement the defects due to chromosomal uvrB deletions, which extend from the bioB gene to the chIA locus (Pannekoek et al, 1978). The maximal protein coding capacity of the cloned region (1920 bp) on pNP5 DNA corresponds with a polypeptide of about 70 000 daltons.…”

“…We have employed the technique of insertional inactivation, using transposons Tnl and Tn5 coding for, respectively, Apr and Km r, to determine the position of the cloned E. coli uvrB locus on a recombinant plasmid, denoted pNP5 (Pannekoek et al, 1978). Plasmid pNP5 consists of the vector pMB9 and an EcoRI fragment, harbouring the uvrB locus, which has been inserted into the unique EcoRI site of the vector.…”

The structure and function of the regulatory elements of the Escherichia coli uvrB gene

“…Digestion of plasmid DNA with restriction endonucleases was done as prescribed by the enzyme manufacturers. Ligation of DNA fragments containing cohesive ends and transformation of competent cells was carried out as described before (10). Initial characterization of a UV resistant phenotype of transformants of strain HP3435 (AuvrB) was done by streaking single colonies 2 and irradiation of a segment of the plates with a UV dose of 150 erg/mm2.…”