REGULATION OF FLAVIN-MONOOXYGENASE GENE EXPRESSION

黄素单加氧酶基因表达的调控

• 批准号: 2095186
• 负责人: RONALD N HINES
• 金额: $ 14.28万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-05 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095186
• 关键词: cytochrome P450; detoxification; drug metabolism; flavins; gender difference; gene expression; genetic library; genetic transcription; gestational age; hormone regulation /control mechanism; human subject; isozymes; laboratory rabbit; liver; lung; molecular cloning; nuclear runoff assay; nucleic acid sequence; oxygenases; posttranscriptional RNA processing; restriction fragment length polymorphism; steroid hormone

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.

猪肝细胞色素P450非依赖性单加氧酶活性， 1964年首次鉴定，随后进行了纯化和鉴定。 这种酶是一种独特的含黄素单加氧酶（FMO）， 细胞色素P450在细胞凋亡中的作用， 代谢活化的各种氮，硫，和 含磷异生物素，包括发现的芳基胺和烷基胺 在香烟烟雾冷凝物和膦酸酯类杀虫剂中。 作为 因此，这种酶被认为在早期 化学致癌和毒性事件。 FMO是一个积分 膜蛋白定位于内质网和细胞核。 两个研究小组独立报告说，两个，也许作为 存在多达四种相关的FMO同工酶。 一种主要的同工酶 主要在肺中发现，而另一种主要在 肝脏 这两种酶似乎都受到激素和个体发育的控制。 最近，已经分离出两个兔cDNA克隆，代表了 FMO的主要肝和肺形式。 这两种mRNA共享56% 序列同一性并表现出组织特异性表达模式。 采用逆转录酶/PCR，这两种cDNA也被 在私家侦探的实验室里分离克隆出来的 的研究目标 目前的建议如下：第一，表达肺 肝脏同工酶将作为组织、性别、胎儿 母鹿的发育和妊娠时间。 第二， 转录或转录后控制将通过以下方式来回答： 在分离的细胞核中通过径流测定来检查转录速率。 第三，FMO同工酶的表达，特别是响应于 类固醇治疗，将在建立的人细胞系中进行检查。 第四，人类细胞系的基因。 第四， 人和兔FMO同工酶将被分离和表征， 限制性内切酶图谱和DNA序列分析。 第五、 FMO基因上的特异性顺式调节序列将通过 与异源报告基因的融合和瞬时表达测定。 最后，哺乳动物和酵母异源表达系统将被研究。 开发 酵母系统将用于回答有关 使用嵌合的FMO同工酶的结构/活性关系 构建和定点诱变。 虽然超出了范围 根据这一建议，哺乳动物系统将有助于确定 FMO系统激活特定原致癌物的能力和/或 原毒素，以及测试这种激活的后果。