Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters.
Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.
Nuyts
S
S
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium. Sandra.Nuyts@rega.kuleven.ac.be
Van Mellaert
L
L
Barbé
S
S
Lammertyn
E
E
Theys
J
J
Landuyt
W
W
Bosmans
E
E
Lambin
P
P
Anné
J
J
D000818:Animals; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D003013:Clostridium / Q000235:genetics / Q000528:radiation effects*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000528:radiation effects*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000528:radiation effects*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013014:SOS Response, Genetics / Q000235:genetics / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D014409:Tumor Necrosis Factor-alpha; D011938:Rec A Recombinases; D002480:Cellulase