Phage Trojan horses: a conditional expression system for lethal genes.
The EcoRI restriction enzyme (ENase) cleaves DNA molecules within the sequence GAATTC. Cells expressing this lethal activity normally make a second enzyme, the M.EcoRI methyltransferase (MTase), which protects their chromosomal DNA by modifying the EcoRI recognition sites. To isolate mutants of the EcoRI ENase, its gene was cloned into a filamentous phage vector (M13mp18) under control of the lac promoter. Normally, filamentous phages (M13, f1 and their derivatives) form turbid plaques by impairing the growth of their host cell without killing it. In contrast, phages expressing the EcoRI ENase kill the host cell, but survive long enough to produce plaques which are very clear. Expression of the M.EcoRI MTase rescues the host and restores turbid plaque formation. EcoRI ENase mutants were isolated by screening for mutants that make turbid, instead of clear, plaques on an M- host. This conditional expression system may be useful for cloning and mutating genes for other toxic proteins.
Heitman
J
J
Rockefeller University, New York, NY 10021.
Fulford
W
W
Model
P
P
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003090:Coliphages / Q000187:drug effects / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005804:Genes, Lethal; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation
D007544:Isopropyl Thiogalactoside; D015246:Deoxyribonuclease EcoRI