Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.

Abstract

Endo.cntdot.R.cntdot.HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E. coli K-12. The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele. These transformants became sensitive to UV light and recombination deficient, and showed a 25-fold increase in the level of exonuclease I activity. The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity were also determined in wild-type and recA1 genetic backgrounds. The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and a 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant may be a result of increased plasmid stability in this genetic background.