Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae
Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae
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DOI：
10.1002/(sici)1098-2280(1996)27:1
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发表时间：
1996
影响因子：
2.8
通讯作者：
Catherine Saner;B. Weibel;F. Würgler;C. Sengstag
中科院分区：
文献类型：
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作者：
Catherine Saner;B. Weibel;F. Würgler;C. Sengstag
黑腹果蝇的体细胞突变和重组试验（SMART）允许在多细胞生物体中筛选化学品的遗传毒性。为了将SMART中获得的数据与啮齿动物遗传毒性试验中获得的数据相关联，重要的是要了解更多关于该昆虫中存在的各种药物代谢酶的信息，并确定其底物特异性。在这项研究中，我们集中在I相酶细胞色素P450 6A 2，这是第一个细胞色素P450从果蝇克隆。对CYP 6A 2基因组DNA片段及其相应的cDNA进行了克隆和测序，发现了一个先前未鉴定的内含子，其具有一个框内终止密码子。该内含子恒定地存在于杀虫剂抗性[OR（R）]和敏感（flr 3）菌株中。发育北方分析表明，CYP 6A 2 mRNA的表达在第三幼虫和蛹阶段的高峰。发现CYP 6A 2 mRNA在杀虫剂抗性菌株中的水平高于杀虫剂敏感菌株。因此，杀虫剂抗性可能与CYP 6A 2表达增强相关。通过在酵母酿酒酵母中共表达CYP 6A 2 cDNA与人NADPH-细胞色素P450还原酶的cDNA，研究了CYP 6A 2酶的底物特异性。转化菌株将真菌毒素黄曲霉毒素B1激活为诱导基因转化的产物，在trp 5位点进行评分。另外两种化合物，7，12-二甲基苯并-[a]蒽（DMBA）和3-氨基-1-甲基-5H-吡啶并[4，3-B]吲哚（Trp-P-2）在转化菌株中代谢为细胞毒性产物。© 1996 Wiley利斯公司
The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug‐metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr3) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide‐resistant strain at higher levels than in the insecticide‐sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH‐cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B1 to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7, 12‐dimethylbenz‐[a]anthracene (DMBA) and 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐2), were metabolized in the transformed strain to cytotoxic products. © 1996 Wiley‐Liss, Inc.