Open AccessAmplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.
Author(s) -
Daniel Vapnek,
N. K. Alton,
Carole L. Bassett,
Sidney R. Kushner
Publication year - 1976
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.73.10.3492
Subject(s) - plasmid , biology , exonuclease , restriction enzyme , escherichia coli , cole1 , hindiii , genetics , microbiology and biotechnology , hybrid plasmid , mutant , endonuclease , exonuclease iii , ultraviolet light , dna , gene , chemistry , dna polymerase , photochemistry
Endo-R-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 ultraviolet light and recombination defieient 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 have also been 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 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background.