Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene

Robert (Stephen) Lloyd, Mary Lou Augustine

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 % of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.

Original languageEnglish (US)
Pages (from-to)89-100
Number of pages12
JournalMutation Research DNA Repair Reports
Volume165
Issue number2
DOIs
StatePublished - 1986
Externally publishedYes

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gene fusion
bacteriophages
molecular cloning
galactosidases
genes
proteins
molecular weight
Escherichia coli
antibodies
DNA libraries
pyrimidines
polyclonal antibodies
rabbits
clones
DNA
assays
enzymes

Cite this

Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene. / Lloyd, Robert (Stephen); Augustine, Mary Lou.

In: Mutation Research DNA Repair Reports, Vol. 165, No. 2, 1986, p. 89-100.

Research output: Contribution to journalArticle

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abstract = "Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 {\%} of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.",
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