| Abstract: | The structure and function of recombinant plasmid pNP5, which consists of vector pMB9 and a 2.5 kb EcoRI fragment harbouring the Escherichia coli uvrB gene, has been investigated. Insertional inactivation with the transposons Tn1 (Apr) or Tn5 (Kmr) has been used to determine the region on pNP5 DNA that is essential for UV resistance in uvrB deletion strains. This region spans approx. 1.8 kb and is separated by at least 280 bp from the pMB9 promoter to which it has been fused. Furthermore, a procedure is described to eliminate the polarity exerted by the transposon Tn5. A combination of in vitro digestion of pNP5::Tn5 DNA with restriction endonuclease XHoI, followed by ligation and subsequent in vivo propagation of the resulting plasmid DNA yields predominantly pNP5 molecules with a site-specific nonpolar mutation. The method allows an investigation of cloned complex genetic units, such as operons. |
