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

R. Stephen Lloyd, Mary Lou Augustine

Research output: Contribution to journalArticle

7 Scopus citations

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 - Mar 1986

ASJC Scopus subject areas

  • Medicine(all)

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