A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene.
A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec.
Yazynin
S A
SA
Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, Moscow.
Deyev
S M
SM
Jucovic
M
M
Hartley
R W
RW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D005804:Genes, Lethal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D012260:Ribonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease