TY - JOUR
T1 - Highly sensitive detection of cytotoxicity using a modified HSP70B′ promoter
AU - Wada, Ken Ichi
AU - Taniguchi, Akiyoshi
AU - Okano, Teruo
PY - 2007/7/1
Y1 - 2007/7/1
N2 - We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B′ gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B′ promoter. 5′- and 3′-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B′ gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B′ gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B′ promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B′ promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B′ promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.
AB - We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B′ gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B′ promoter. 5′- and 3′-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B′ gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B′ gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B′ promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B′ promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B′ promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.
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U2 - 10.1002/bit.21293
DO - 10.1002/bit.21293
M3 - Article
C2 - 17163517
AN - SCOPUS:34447339101
VL - 97
SP - 871
EP - 876
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 4
ER -