Cloning, expression and stable maintenance of a bacteriophage DNA repair gene in E. coli
Efforts to clone the bacteriophage T4 DNA repair gene, denV, have met with repeated problems of gene instability. Therefore, oligonucleotide site-directed mutagenesis was used to create new DNA restriction sites closely flanking the structural gene, facilitating the removal of adjacent deleterious T4 sequences. The sequence which encodes the DNA repair enzyme, endonuclease V, was then reconstructed behind the hybrid lambda promoter O/sub L/P/sub R/ and its associated ribosome-binding site in a plasmid vector for expression in E. coli. Transformants harboring the expression plasmid bearing the correct reconstruction of denV were screened and selected by colony hybridization to a synthetic bridge oligonucleotide and by restriction analyses. The denV structural gene was found inserted at an equal frequency in both orientations relative to the promoter. These plasmids were transformed into an E. coli strain which is defective for UV excision recombination and repair (uvrA/sup -/, recA/sup -/). The defective hosts were assayed for survival after UV irradiation, and expression of the denV gene was found to enhance UV survival to levels indicative of full complementation of the uvrA/sup -/ deficiency. Correspondingly, Western blot analyses of cellular protein extracts from the defective host harboring the denV/sup +/ plasmid showed a protein which was immunoreactive with an anti-endonuclease V antibody. Additionally, in infections using a UV-irradiated T4 mutant (T4 denV1, defective for active endonuclease V), these cells fully complemented the mutant phage to wild type survival levels.
- Research Organization:
- Vanderbilt Univ., Nashville, TN
- OSTI ID:
- 6905843
- Report Number(s):
- CONF-8606151-; TRN: 87-004002
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ESCHERICHIA COLI
DNA REPAIR
GENETIC RADIATION EFFECTS
GENES
CLONING
BACTERIOPHAGES
NUCLEOTIDES
PLASMIDS
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CELL CONSTITUENTS
ELECTROMAGNETIC RADIATION
GENETIC EFFECTS
MICROORGANISMS
ORGANIC COMPOUNDS
PARASITES
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
VIRUSES
560130* - Radiation Effects on Microorganisms