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송어 CYP1A유전자 발현조절에서 5'-flanking DNA의 역할

송어 CYP1A유전자 발현조절에서 5'-flanking DNA의 역할
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대학원 약학과
이화여자대학교 대학원
송어 cytochrome P45O 1A 유전자 발현 조절 기전에 관해 조사하기 위하여 송어 cytochrome P45O 1A 의 5'-flanking DNA를 repoter 유전자인 CAT 의 promoter 앞에 연결하였다. 송어 CYP1A1-CAT construct를 Hepa-1세포에 transfection한 다음 다양한 약물을 단독 또는 3MC와 병용 투여하였다. 24시간 동안의 약물 처리 후에 각 약물에 의해 발현되는 CAT mRNA 양을 RT-PCR을 이용하여 측정하였다. CAT mRNA는 3MC 농도별, 시간별 처리에 따라 대조군에 비해 1.03∼2.08 배 증가하였고, 이렇게 증가된 CAT mRNA는 3MC와 동시 처리된 dexamethasone, methoxsalen, retinoic acid, estradiol과 3MC를 가하기12시간 전에 처리된 morin등에 의해 감소되었다. 그 정도는 3MC 단독 처리를 100%로 보았을 때 각각 92∼66%, 91∼64%, 95.7∼55.3%, 85.6∼59.6%, 43.8%를 나타내었다. 송어 cytochrome P450 1A 유전자의 5'-flanking부위에 유전자 발현을 저해하는 인자나 유도하는 인자들과 반응하는 (negative or positive regulatory) cis-element에 대해 알아보기 위해 송어 CYP1A1 유전자의 5'-flanking 부위를 부분적으로 결손 시킨 construct를 이용한 조사를 실시하였다. 송어 CYP1A1 유전자의 5'-flanking DNA중 -1562∼-1343 bp가 결손된 Δ2H construct가 transfection된 Hepa-I 세포에서는 CAT mRNA의 기초적인 발현 (constitutive level)이 wild type의 경우에 비해 약 2배 정도 증가하였으며, 3MC에 의한 유전자 발현 유도 작용은 관찰되지 않았다. 또, -3400∼-1350 bP가 결손된 Δ3H construct 의 경우에는 3MC에 의한 발현 유도 작용 및 morin과 3MC 병용 처리에 의한 발현 저해 작용이 모두 관찰되지 많았다. 이러한 결과를 통해 저해 인자와 반응하는 cis-element가 5'-flanking DNA중 -1562∼-1350 bp에 존재함을 알 수 있었고, 이 부위가 유도 인자들과 반응하는 cis-element와도 상호 작용을 할 수도 있음을 추정할 수 있었다.;In order to gain insight into the mechanism of regulation of trout CYP1A1 gene expression, the 5'-flanking region of a trout CYP1A1 was cloned into CAT-basic expression vector, This trout CP1A1-CAT construct was transfected into Hepa-Ⅰ cells and various chemicals were treated alone or concomitantly with 3MC. After treatment for 24 hours, the expression of CAT mRNA by various chemicals assayed via RTPCR. The expression of CAT mRNA by 3MC was increased by 1.03∼2.08 fold in time and concentration dependent manner, as compared with that of control. This increase of CAT mRNA was decreased by concomitantly treated dexamethasone, methoxsalen, retinoic acid, estradiol and pretreated morin 12 hours prior to the addition of 3MC. The level of CAT mRNA were respectively 92∼66%, 91∼64%, 95.7∼55.3%, 85.6∼59.6% and 43.8% of 3MC stimulated CAT mRNA. To find out if there are positive or negative regulatory cis-element in trout CYP1A1 gene, further investigation using deleted trout CYPIAl-CAT construct was undertaken. 3MC or morin+3MC was treated into Hepa-Ⅰ cells transfected with these deleted constructs (Δ2H or Δ3H) and the expression level of CAT mRNA were compared with that of wild type construct. In cells transfected with Δ2H deleted trout CYP1A1-CAT construct that was deleted in -1562∼-1350 bp of trout CYP1A1 5'-flanking region, the constitutive expression of CAT mRNA was about two fold higher than in cells transfected with wild type construct and the stimulation effect of 3MC was no longer observed. In the cells transfected with the Δ3H deleted construct which was deleted in -3400∼1350 bp, both the 3MC stimulation and the morin inhibition were not observed. These results of the deletion study suggested that the negative regulatory cis-element exist in -1562∼-1350 bp of trout CYP1A1 5'-flanking region and in some ways, this negative element might interact with the positive regulatory element to modulate the expression of CYP1A1 by 3MC.
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