REGULATION OF GENE EXPRESSION BY DIETARY FAT

这项工作的长期目标是了解分子 膳食多不饱和脂肪抑制基因表达的机制。 美国人被鼓励消耗更少的脂肪和更高的多不饱和脂肪酸。 饱和脂肪的比例。这一建议的饮食目标是预防 由于脂肪摄入量、血清脂质 和患心脏病的风险。对细胞内的 由于改变饮食中脂肪的类型和数量而导致的事件。 多不饱和脂肪酸在生物体内有无数的作用 包括许多基因的转录调控。然而，在这方面， 脂肪酸改变基因表达的机制仍然是 测定葡萄糖-6-磷酸脱氢酶（G6 PD）被抑制， 膳食中的多不饱和脂肪和多不饱和脂肪酸 培养的肝细胞。因此，G6 PD将被用作我们的模型基因。 本申请中描述的研究计划将确定 多不饱和脂肪酸对G6 PD的抑制作用发生在 转录后步骤，然后确定分子基础， 这种抑制发生。在特定目标1中，基因组DNA探针 将被表征并产生cDNA用于分析 转录活性和mRNA积累。仅适用于空闲的探头 重复的元素和独特的，就其出现在 将使用基因组。在具体目标2中， 转录1与转录后过程对调节 将通过膳食脂肪测定G6 PD。老鼠被喂食高葡萄糖，低脂肪 饮食将与喂食补充有以下物质的高葡萄糖饮食的小鼠进行比较 红花油转录活性将使用核转录因子来测量。 连续试验，并与G6 PD mRNA积累的变化进行比较 和酶的活性。在具体目标3中， 哪种脂肪酸抑制肝细胞中G6 PD的表达， 测定转录活性、mRNA丰度和酶活性 将在小鼠肝细胞+/-亚油酸酯和 花生四烯酸在具体目标4中， 将测定脂肪酸的G6 PD表达。如果调控 主要是转录，顺式作用的DNA元件在G6 PD 5 '- 侧翼DNA将使用DNase I超敏性测定来鉴定， 缺失突变体和体外DNA的功能转染分析 结合测定。如果调控是转录后的， mRNA序列中的顺式作用元件 将使用G6 PD RNA的功能转染分析来鉴定 与报告基因相连的序列。在这两种情况下， 元素的特征在于其赋予脂肪酸 在异源启动子或基因上的调节，并且将被定义为 序列特异性突变。

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.