Cloning, expression and stable maintenance of a bacteriophage DNA repair gene in E. coli

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6905843

Recinos, A III; Lloyd, R S

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-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

560130* -- Radiation Effects on Microorganisms
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
BACTERIA
BACTERIOPHAGES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CELL CONSTITUENTS
CLONING
DNA REPAIR
ELECTROMAGNETIC RADIATION
ESCHERICHIA COLI
GENES
GENETIC EFFECTS
GENETIC RADIATION EFFECTS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
PARASITES
PLASMIDS
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
ULTRAVIOLET RADIATION
VIRUSES