AFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

描述：(改编自申请者摘要)寄生曲霉 和相关真菌经常在食物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 肝毒性、诱变性、致畸性、免疫毒性和是最大的 某些动物体内已知的天然致癌物质。数据也 强烈提示黄曲霉毒素B_1是原发性肝损害的危险因素 人类的癌症。这项研究的长期目标是消除黄曲霉毒素B 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 黄曲霉毒素B_1的生物合成。拟议的研究旨在确定几个 AFB1生物合成途径中提供靶点的控制点 由产生或引入其上的化合物抑制毒素合成 寄主植物。 提出了以下具体目标，以深入开展 从分子水平上理解基因表达的调控机制 转录和蛋白质定位。1.确定特定的顺位代理 调节调节的位点和反式作用调节因子(TAF) 黄曲霉毒素B_1的生物合成。2.分析亚细胞定位和 催化晚期通路功能的酶的相互作用 黄曲霉毒素B_1合成。要实现特定目标1，凝胶迁移率漂移和DNA酶I 足迹分析将绘制顺式作用地点在 参与AFB1合成的关键基因的启动子。功能界别 这些部位的意义将在活体内通过测量 定向诱变对β-葡萄糖苷酶表达的影响 (GUS)记者在适当的生长条件下建造。基因 编码TAF将使用现有的技术和影响进行克隆 营养、环境因素和真菌发育对TAF的影响 将分析表达/活动。实现特定目标2，多克隆 抗体将被用来定位参与AFB1合成的关键酶 通过细胞分离、免疫定位和电子显微镜观察。这些 同样的方案加上免疫沉淀将被用来研究 真菌中这些酶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.