REGULATION OF FLAVIN-MONOOXYGENASE GENE EXPRESSION

猪肝细胞色素P450不依赖于细胞色素P450的单加氧酶活性是 1964年首次鉴定，随后进行了提纯和表征。 这种酶是一种独特的含有黄素的单加氧酶(FMO)，它发挥着 一个与细胞色素P450同等重要的角色，如果不是更重要的话 多种氮、硫和硫的代谢激活 发现含磷的外来生物，包括芳基胺和烷基胺 香烟烟雾中的冷凝物和磷酸盐类杀虫剂。AS 因此，这种酶被认为在早期 化学致癌和毒性事件。FMO是一个整体 膜蛋白定位于内质网和胞核。 两个研究小组已经独立报告了这两个，也许是 有多达四种与FMO相关的同工酶。一种主要的同工酶 主要存在于肺部，而另一种主要存在于 肝脏。这两种酶似乎都受到荷尔蒙和个体基因的控制。 最近，已经分离到两个代表兔的cdna克隆。 主要的肝和肺形式的FMO。这两个mRNAs共享56% 序列同一性和组织特异性表达模式。 利用逆转录酶/聚合酶链式反应，这两个cDNA也都被 在PI的实验室中分离和克隆。的研究目标 目前的建议如下：第一，肺的表现 肝脏同工酶将作为组织、性别、胎儿的功能进行检查 母鹿的发育和受孕时间。第二，关于 转录或转录后控制将通过以下方式回答 在分离的细胞核中用径流分析法检测转录速率。 第三，FMO同工酶的表达，特别是在对 类固醇治疗，将在已建立的人类细胞系中进行检验。 第四，人类细胞系的基因。第四，人类的基因 人和兔的FMO同工酶将被分离并鉴定为 限制性内切酶图谱和DNA序列分析。第五, FMO基因上的特定顺式调控序列将通过 与异源报告基因融合和瞬时表达分析。 最后，哺乳动物和酵母的异源表达系统将 发展起来的。酵母系统将用于回答以下问题 用嵌合体方法研究FMO同工酶的结构/活性关系 构建和定点突变。虽然超出了范围 这一建议中，哺乳动物系统将有助于确定 FMO系统激活特定致癌物和/或的能力 原毒素，以及测试这种激活的后果。

A cytochrome P450-independent monooxygenase activity in porcine liver was first identified in 1964 and subsequently purified and characterized. This enzyme is a unique flavin-containing monooxygenase (FMO) which plays an equal, if not more important role that cytochrome P450 in the metabolic activation of a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, including aryl- and alkylamines found in cigarette smoke condensates and phosphonate type insecticides. As such, this enzyme is thought to play an important role in the early events of chemical carcinogenesis and toxicity. The FMO is an integral membrane protein localized both in the endoplasmic reticulum and nucleus. Two research groups have reported independently that two, and perhaps as many as four related isozymes of FMO exist. One of the major isozymes is found predominantly in the lung while the other predominantly in the liver. Both enzymes appear to be under hormonal and ontogenic control. Recently, two rabbit cDNA clones have been isolated representing the major hepatic and pulmonary forms of FMO. The two mRNAs share 56% sequence identity and exhibit tissue-specific expressions patterns. Employing reverse transcriptase/PCR, both of these cDNAs have also been isolated and cloned in the PI's laboratory. The research objectives of the current proposal are as follows: first, the expression of the lung and liver isozymes will be examined as a function of tissue, sex, fetal development and time of gestation in the doe. Second, the question of transcriptional or post-transcriptional control will be answered by examining the rate of transcription by run-off assays in isolated nuclei. Third, the expression of the FMO isozymes, particularly in response to steroid treatment, will be examined both in established human cell lines. Fourth, the genes for the human cell lines. Fourth, the genes for the human and rabbit FMO isozymes will be isolated and characterized by restriction endonuclease mapping and DNA sequence analysis. Fifth, specific cis-regulatory sequences on the FMO genes will be identified by fusion with a heterologous reporter gene and transient expression assays. Finally, mammalian and yeast heterologous expression systems will be developed. The yeast system will be used to answer questions regarding structure/activity relationships for the FMO isozymes using chimeric constructions and site-directed mutagenesis. Although beyond the scope of this proposal, the mammalian system will be useful in determining the ability of the FMO system to activate specific procarcinogens and/or protoxins, as well as testing the consequences of that activation.