AFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

描述：（改编自申请人的摘要）寄生曲霉 和相关真菌经常在食物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（AFB 1）I 肝毒性、致突变性、致畸性、免疫毒性，并且是最常见的 在某些动物中已知的强效天然致癌物。 数据还 这强烈表明AFB 1是原发性肝脏的一个危险因素 人类的癌症 这项研究的长期目标是消除AFB 1。 从食物链中。 短期目标是了解 调节参与免疫反应的关键基因表达的机制 AFB 1的合成。 拟议的研究旨在确定几个 AFB 1生物合成途径中的控制点， 通过由其产生或引入其上的化合物抑制毒素合成 寄主植物 建议以下具体目标，以制定一项深入的 了解调节基因表达的机制， 转录和蛋白质定位。 1. 确定特定的顺式作用 位点和介导调节的反式作用调节因子（TAF） AFB 1的生物合成。 2. 分析亚细胞定位， 参与的催化晚期途径功能的酶的相互作用 黄曲霉毒素B1合成。 为了实现具体目标1，凝胶迁移率改变和DNA酶I 足迹分析将绘制顺式作用位点的位置， 参与黄曲霉毒素B1合成的关键基因的启动子。 功能 这些位点的显著性将通过测量 定向诱变对β-葡糖苷酶表达的影响 (GUS)报告基因构建体在适当的生长条件下。 基因 编码TAF将使用已建立的技术进行克隆， 营养物质、环境因素和真菌发育对TAF的影响 将分析表达/活性。 为了实现特定目标2，多克隆 抗体将用于定位参与黄曲霉毒素B1合成的关键酶 通过细胞分级、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

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.