Expression of the cloned uvrB gene of Escherichia coli: mode of transcription and orientation

Abstract

The Escherichia coli uvrB gene, located on a 1.5-megadalton EcoRI (fragment F, derived from transducing phage lambda b2att2 [lambda b2cI857intam6 delta (bioAB)bio-FCD+uvrB+], has been cloned in the unique EcoRI site of several "relaxed" plasmids, i.e., pMB9, pBR322, and pBH20 (= ;BR322, including the lac regulatory elements [K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F. Bolivar, and H. W. Boyer, Science 198:1056--1063, 1977]y. Expression of the uvrB gene, both on pMB9 and on pBH20, occurs only when fragment F has one particular orientation. Cloning of this fragment on pBR322 in either orientation does not allow expression of the uvrB gene. Transcription of this gene on pNP5 ( = pMB9 uvrB) is shown to be dependent on a pMB9 promotor that is located on a 0.22-megadalton EcoRI-HindIII fragment. Using plasmid pBH20 as a vector, we could demonstrate that expression of the uvrB gene is under control of the lac promotor-operator region. From deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments with lambda pgal8 deoxyribonucleic acid and restriction fragments of pNP5 deoxyribonucleic acid it could be shown that the uvrB gene is transcribed clockwise on the chromosome.