The Drosophila cytochrome P450 gene Cyp6a2: Structure, localization, heterologous expression, and induction by phenobarbital
The Drosophila cytochrome P450 gene Cyp6a2: Structure, localization, heterologous expression, and induction by phenobarbital
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DOI：
10.1089/dna.1997.16.1345
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发表时间：
1997-11-01
影响因子：
3.1
通讯作者：
Feyereisen, R
中科院分区：
文献类型：
--
作者：
Dunkov, BC;Guzov, VM;Feyereisen, R
黑腹果蝇细胞色素P450基因Cyp 6a 2位于2号染色体右臂43 A1 -2位，由两个外显子组成，外显子之间有一个69 bp的内含子。用苯巴比妥处理果蝇后，CYP 6A 2 mRNA水平迅速升高，CYP 6A 2蛋白产量增加。当CYP 6A 2启动子区的DNA与荧光素酶报告基因连接并转染果蝇后，CYP 6A 2启动子区的DNA具有功能。此外，苯巴比妥对荧光素酶活性的剂量依赖性诱导表明，苯巴比妥诱导所需的元件位于翻译起始位点的428 bp内。通过细胞裂解物的Western印迹和P450的还原CO复合物的光谱表征，观察到CYP 6A 2蛋白在感染Cyp 6a 2重组杆状病毒的鳞翅目细胞中的异源表达。在该系统中产生的CYP 6A 2蛋白将艾氏剂和七氯代谢为它们的环氧化物，并通过脱硫将杀虫剂二嗪农代谢为二唑磷，通过氧化酯裂解将其代谢为2-异丙基-4-甲基-6-羟基嘧啶。通过添加纯化的家蝇NADPH细胞色素P450还原酶和细胞色素B，重组杆状病毒感染的细胞裂解物中的代谢大大增强（5）。这些结果表明CYP 6A 2催化有机磷杀虫剂的代谢，并且它们暗示Cyp 6a 2过表达与代谢抵抗有关。Cyp 6a 2基因似乎是一个合适的模型，用于苯巴比妥诱导过程的遗传分析。
The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein, DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells, Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b(5). These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.