Construction of artificial promoters sensitively responsive to sonication in vitro
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To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro.
The binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay.
Of 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm2 for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication.
A promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.
Keywordspromoter transcription oxidative stress
- 21.Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001.Google Scholar