REGULATION OF GENE EXPRESSION BY DIETARY FAT

这项工作的长期目标是理解分子 膳食多不饱和脂肪抑制基因表达的机制。 鼓励美国人少吃脂肪，多吃多不饱和脂肪 与饱和脂肪的比例。这个推荐的饮食目标是预防性的。 健康量度因脂肪摄入量与血脂的相关性 以及患心脏病的风险。人们对细胞内的 由于改变饮食中脂肪的类型和数量而引起的事件。 多不饱和脂肪酸在生物体内有无数种作用。 包括一些基因的转录调控。然而， 脂肪酸改变基因表达的机制尚不清楚 下定决心。葡萄糖-6-磷酸脱氢酶(G6PD)被抑制 膳食中的多不饱和脂肪和多不饱和脂肪酸 培养中的肝细胞。因此，G6PD将作为我们的模型基因。 本申请中描述的研究计划将决定 多不饱和脂肪酸对G6PD的抑制作用发生在 转录后步骤，然后通过 这种抑制就会发生。在特定目标1中，基因组DNA探针 将对其进行表征并生成用于分析 转录活性和信使核糖核酸的积累。只有免费的探头 具有重复性的元素，并且就它们在 基因组将被使用。在具体目标2中， 转录因子1与转录后过程在调控中的作用 G6PD受膳食脂肪影响将被测定。小鼠喂食高糖、低脂肪 饮食将被比作喂食高糖饮食并补充 红花油。转录活性将用核来测量 Run-On法并与G6PD mRNA积聚变化进行比较 和酶的活性。在特定目标3中，分子水平在 哪些脂肪酸会抑制肝细胞中G6PD的表达 下定决心。转录活性、信使核糖核酸丰度和酶活性 将在原代培养的小鼠肝细胞/-亚油酸和 花生四烯酸。在特定的目标4，抑制的分子基础 将测定脂肪酸对G6PD的表达。如果监管是 主要是转录方面，G6PD 5‘中的顺式作用DNA元件- 侧翼DNA将通过DNase I超敏分析进行鉴定， 缺失突变体和体外DNA的功能转染分析 有约束力的分析。如果监管是转录后的，那么准确的步骤是 将被定位，并且顺式作用元件在mRNA序列中 将通过G6PD RNA的功能转染分析进行鉴定 与报告基因相关联的序列。在这两种情况下，推定的反应 元素将以其提供脂肪酸的能力为特征 对异源启动子或基因的调控，并将由 序列特定的突变。

The long-term objective of this work is to understand the molecular mechanism by which dietary polyunsaturated fats inhibit gene expression. Americans are encouraged to consume less fat and a higher polyunsaturated to saturated fat ratio. This recommended dietary goal is a preventive health measure because of the correlation between fat intake, serum lipids and the risk of heart disease. Little is known about the intracellular events resulting from changing the type and quantity of fat in the diet. Polyunsaturated fatty acids have a myriad of actions within the organism including the transcriptional regulation of a number of genes. However, the mechanisms by which fatty acids alter gene expression remain to be determined. Glucose-6-phosphate dehydrogenase (G6PD) is inhibited both by dietary polyunsaturated fats and by polyunsaturated fatty acids in hepatocytes in culture. Consequently, G6PD will be used as our model gene. The research program described in this application will determine if the inhibition of G6PD by polyunsaturated fatty acids occurs at a pre- or post-transcriptional step and then to determine the molecular basis by which this inhibition occurs. In Specific Aim 1, the genomic DNA probes will be characterized and a cDNA generated for the analysis of transcriptional activity and mRNA accumulation. Only probes which are free of repetitive elements and unique with respect of their occurrence within the genome will be used. In Specific Aim 2, the relative importance of transcriptiona1 versus post-transcriptional processes on the regulation of G6PD by dietary fat will be determined. Mice fed a high glucose, low fat diet will be compared to mice fed a high glucose diet supplemented with safflower oil. Transcriptional activity will be measured using the nuclear run-on assay and compared with changes in the accumulation of G6PD mRNA and activity of the enzyme. In Specific Aim 3, the molecular level at which fatty acids inhibit G6PD expression in hepatocytes will be determined. Transcriptional activity, mRNA abundance and enzyme activity will be compared in primary cultures of mouse hepatocyte+/- linoleate and arachidonate. In Specific Aim 4, the molecular basis for the inhibition of G6PD expression by fatty acids will be determined. If regulation is primarily transcriptional, the cis-acting DNA element in the G6PD 5'- flanking DNA will be identified using DNase I hypersensitivity assays, functional transfection analyses of deletion mutants and in vitro DNA binding assays. If regulation is post-transcriptional, the precise step will be localized and the cis-acting element within the mRNA sequences will be identified using functional transfection analyses of G6PD RNA sequences linked to a reporter gene. In both cases the putative response element will be characterized for its ability to confer fatty acid regulation on a heterologous promoter or gene and will be defined by sequence specific mutations.