NUTRITIONAL AND HORMONAL REGULATION OF HEPATIC GENES

这项建议的目的是研究黄体病毒感染的分子机制。 甲状腺激素和营养因素在调节特异性中的作用 肝脏中的mRNA水平。重点将放在确定是否 调控发生在基因转录水平，如果是这样的话， 开发一种识别控制所必需的DNA元件的系统。 支持生物合成所需的还原型NADPH的产生 肝脏中的过程主要由三种酶催化--苹果酸 葡萄糖-6-磷酸脱氢酶(G6PD)和6-磷酸葡萄糖酸 脱氢酶(6PGD)。这三种酶的肝脏含量是 以类似的方式调节：甲亢或甲亢后升高 喂食促脂饮食，糖尿病、饥饿或抑郁 高血糖素血症。以前的研究已经证明，调控是存在的 主要是在翻译前水平上改变细胞内的mRNA水平 每种酶。因此，可以想象，这3种NADPH依赖 脱氢酶在大鼠肝脏中是一个协调调控的家族。至 进一步研究调控，获得与mRNA同源的特异性cDNA克隆 每种酶的编码都将被分离。这些cdna探针将允许 用分子生物学方法灵敏、准确地检测信使核糖核酸 杂交技术。甲状腺激素或饮食因素的影响 苹果酸酶、G6PGD和G6PGD基因的核转录速率 将会受到考验。与这些酶相对应的基因组DNA将是 从大鼠DNA文库中分离出基因并与基因拷贝进行比较 5个插入序列和DNA序列的数目、数目和位置 脚-侧翼区域。最后，分离的基因的功能将是 通过将其导入含有受体的完整培养细胞中进行测试 甲状腺激素和评估mRNA是否通过转录产生 这些基因在体内。如果是这样，甲状腺激素的调节能力 新引入的DNA的mRNA的表达将通过 媒体中的甲状腺激素水平各不相同。这样一种分析方法的发展 系统将允许定义DNA序列的范围和性质 对使用‘体外’遗传学技术进行调控是必不可少的。这 系统将提供一个很好的机会来研究实际的机制 荷尔蒙和营养因素可以通过这种方式改变基因表达。

The goal of this proposal is to investigate the molecular mechanism of action of thyroid hormone and nutritional factors in modulating specific mRNA levels in the liver. Emphasis will be focused on determining whether regulation occurs at the level of gene transcription and, if so, to developing a system for identifying the DNA elements essential to control. The generation of reduced NADPH necessary for supporting biosynthetic processes in the liver is catalyzed primarily by three enzymes - malic enzyme, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The hepatic content of these three enzymes is regulated in a similar fashion: elevated in hyperthyroidism or after feeding lipogenic diet and depressed in diabetes, starvation or hyperglucagonemia. Previous work has demonstrated that regulation occurs primarily at a pretranslational level to alter cellular levels of mRNA for each enzyme. Thus, it is conceivable that these 3 NADPH-dependent dehydrogenase represent a coordinately regulated family in rat liver. To further investigate the regulation, specific cDNA clones homologous to mRNA coding for each enzyme will be isolated. These cDNA probes will allow a sensitive and accurate measurement of mRNA levels by molecular hybridization techniques. The effect of thyroid hormone or dietary factors on the rate of nuclear transcription of malic enzyme, G6PGD and G6PGD genes will be tested. Genomic DNA corresponding to each of these enzymes will be isolated from a rat DNA library and compared with respect to gene copy number, number and location of intervening sequences and DNA sequences of 5 feet-flanking regions. Finally, the function of isolated genes will be tested by introduction into an intact cultured cell containing receptor for thyroid hormone and assessing whether mRNA is produced by transcription of these gene in vivo. If so, the ability of thyroid hormone to regulate expression of mRNA from the newly introduced DNA will be examined by varying thyroid hormone levels in the media. Development of such an assay system would allow the definition of the extent and nature of DNA sequences essential for regulation using the techniques of 'in vitro' genetics. This system would provide an excellent opportunity to study the actual mechanism by which the hormonal and nutritional factors can modify gene expression.