| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Electrophiles or oxidative stresses induce gene expression of phase 2 detoxification enzymes. In this study, we tried to identify molecular basis of this induction by screening mutant zebrafish that has defects in response to electrophiles. We introduced random mutations in adult male fish using ethylnitrosourea (ENU) as a mutagen. F1 progenies derived from ENU mutagenized fish and subsequent F2 families were raised. Screening of mutant fish was carried out using F3 larvae. F3 larvae at 5 days after fertilization were treated with diethylmaleate (DEM), auranofin (AUR), or control solvent dimethylsulfoxide for 6 hours, and induction of gstp expression in these larvae were analyzed by whole-mount in situ hybridization. We searched for mutant lines which included larvae displaying no or weak induction of gstp after treatment of DEM or AUR. We have screened 125 F2 families and isolated some candidates of mutant lines which had defects in phase 2 induction. We also isolated 70 lines displaying defects in morphogenesis. On the other hands, in order to improve our mutant fish screening, we tried to generate transgenic fish which expresses GFP after treatment of electrophiles. For this purpose, we utilized the gene regulatory region of gstp since it showed the strongest induction after treatment of DEM among phase 2 genes we had tested. By GFP-reporter analysis, we demonstrated that 3 kb region upstream of transcriptional initiation site of gstp was enough for transactivation by Nrf2. We therefore tried to establish stable transgenic lines of containing this gstp-GFP reporter construct in their chromosomes. As a result, three transgenic lines were isolated. Furthermore, we analyzed critical cis-regulatory elements for Nrf2 transactivation in the gstp regulatory region, and finally demonstrated that PARE sequence which was located just 50 bp upstream of transcriptional initiation site was essential for both the transactivation and Nrf2 binding.
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