Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.
Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.
Fuerst
T R
TR
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Fernandez
M P
MP
Moss
B
B
D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007763:Lac Operon; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics*
D004274:DNA, Recombinant; D012097:Repressor Proteins; D014157:Transcription Factors