AFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

寄生曲霉中黄曲霉毒素 B1 的生物合成

• 批准号: 2468698
• 负责人: JOHN E LINZ
• 金额: $ 17.22万
• 依托单位: MICHIGAN 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: 1991-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2468698
• 关键词: Aspergillus; DNA footprinting; aflatoxins; beta glucuronidase; enzyme activity; fungal genetics; gene expression; genetic promoter element; immunoprecipitation; laboratory rabbit; microorganism toxicology; molecular cloning; protein biosynthesis; protein localization; site directed mutagenesis; transcription factor

DESCRIPTION: (Adapted from Applicant's Abstract) Aspergillus parasiticus and related fungi frequently produce aflatoxin contamination in food and feed crops in the US and in other areas of the world. Aflatoxin B1 (AFB1) I hepatotoxic, mutagenic, teratogenic, immunotoxic, and is one of the most potent naturally occurring carcinogens known in certain animals. Data also strongly suggest that AFB1 is a contributory risk factor in primary liver cancer in humans. The long term goal of this research is to eliminate AFB1 from the food chain. The short term goal is to understand the molecular mechanisms which regulate the expression of key genes involved in the biosynthesis of AFB1. The proposed studies are designed to identify several control points in the AFB1 biosynthetic pathway which provide targets for inhibition of toxin synthesis by compounds produced by or introduced onto the host plant. The following specific aims are proposed to develop an in-depth understanding of the mechanisms which regulate gene expression at the level of transcription and protein localization. 1. Identify specific cis-acting sites and trans-acting regulatory factors (TAF) which mediate the regulation of AFB1 biosynthesis. 2. Analyze the subcellular localization and interaction of enzymes which catalyze late pathway functions involved in AFB1 synthesis. To achieve Specific aim 1, Gel mobility shift and DNAse I footprint analyses will map the location of cis-acting sites in the promoters of key genes involved in AFB1 synthesis. The functional significance of these sites will be analyzed in vivo by measuring the effects of directed mutagenesis on the expression of beta-glucuroindase (GUS) reporter constructs under appropriate growth conditions. Genes encoding TAF will be cloned using established technologies and the influence of nutrients, environmental factors, and fungal development on TAF expression/activity will be analyzed. To achieve Specific aim 2, polyclonal antibodies will be used to localize key enzymes involved in AFB1 synthesis by cell fractionation, immunolocalization, and electron microscopy. These same protocols plus immunoprecipitation will be utilized to study the possible physical interaction and co-localization of these enzymes in fungal cells.

描述：（改编自申请人的摘要）寄生曲霉 和相关真菌经常在食品和食品中产生黄曲霉毒素污染 美国和世界其他地区的饲料作物。 黄曲霉毒素 B1 (AFB1) I 具有肝毒性、致突变性、致畸性、免疫毒性，是最严重的毒性之一 在某些动物中已知的强效天然致癌物。 数据还 强烈表明 AFB1 是原发性肝脏的一个促成危险因素 人类癌症。 这项研究的长期目标是消除 AFB1 来自食物链。 短期目标是了解分子 调控关键基因表达的机制 AFB1 的生物合成。 拟议的研究旨在确定几个 AFB1 生物合成途径中的控制点为 通过产生或引入的化合物抑制毒素合成 寄主植物。 为深入发展，提出以下具体目标： 了解在水平上调节基因表达的机制 转录和蛋白质定位。 1. 识别特定的顺式作用 介导调节的位点和反式作用调节因子（TAF） AFB1 生物合成。 2. 分析亚细胞定位和 催化后期通路功能的酶之间的相互作用 AFB1 合成。 为了实现具体目标 1、凝胶迁移率变化和 DNAse I 足迹分析将绘制顺式作用位点的位置 参与 AFB1 合成的关键基因的启动子。 功能性的 通过测量这些位点的重要性将在体内进行分析 定向诱变对β-葡萄糖醛酸苷酶表达的影响 （GUS）记者在适当的生长条件下构建。 基因 编码 TAF 将利用现有技术进行克隆，影响力 TAF 上的营养物质、环境因素和真菌发育 将分析表达/活性。 实现具体目标2，多克隆 抗体将用于定位参与 AFB1 合成的关键酶 通过细胞分级分离、免疫定位和电子显微镜。 这些 将使用相同的方案加上免疫沉淀来研究 这些酶在真菌中可能的物理相互作用和共定位 细胞。