Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5'-flanking regions of the human p21 gene.
A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy gamma-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-kappaB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.
Kagiya
Go
G
School of Allied Health Sciences, Kitasato University, 1-15-1 Sagamihara, Kanagawa 228-8555, Japan.
Ogawa
Ryohei
R
Cook
John A
JA
Choudhuri
Rajani
R
Hatashita
Masanori
M
Tanaka
Yoshikazu
Y
DeGraff
Bill G
BG
Mitchell
James B
JB
D024506:5' Flanking Region / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000528:radiation effects; D011839:Radiation, Ionizing; D016415:Sequence Alignment; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004274:DNA, Recombinant; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D016159:Tumor Suppressor Protein p53