REGULATION OF UREA CYCLE ENZYME SYNTHESIS

尿素循环酶合成的调控

• 批准号: 3231503
• 负责人: SIDNEY MACHEN MORRIS
• 金额: $ 10.64万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3231503
• 关键词: Anura; DNA binding protein; affinity chromatography; binding proteins; carbamoylphosphate synthase; complementary DNA; gene expression; genetic library; genetic manipulation; genetic transcription; hormone regulation /control mechanism; liver metabolism; messenger RNA; molecular cloning; nucleic acid hybridization; nucleic acid sequence; reporter genes; thyroxine; tissue /cell culture; urea cycle

A problem of major interest in current biology centers not only on the question of the mechanisms whereby hormones regulate the expression of specific genes, but also on how coordinate expression of genes -- e.g., for individual enzymes within a metabolic pathway -- is achieved. Expression of the genes encoding enzymes of the urea cycle constitutes an excellent model for investigating both aspects of this problem. The urea cycle is comprised of 2 mitochondrial and 3 cytosolic enzymes, all encoded by nuclear genes which are coordinately expressed to a significant extent only in liver of ureotelic animals. Levels of the urea cycle enzymes in mammals are responsive to changes in dietary protein content, glucocorticoids, glucagon, insulin, and thyroid hormone. Results from this laboratory, as well as from others, have demonstrated that a primary component of these changes involves altered cellular concentrations of the mRNAs for these enzymes. Studies in this laboratory have also demonstrated that the transcription rate of these genes in rat liver is increased in response to cyclic AMP and that glucocorticoids and dibutyryl cyclic AMP are each necessary and sufficient to elevate mRNA levels for urea cycle enzymes in rat hepatocytes cultured in serum-free medium. This grant proposes to isolate and characterize genomic DNA clones encoding two of the urea cycle enzymes -- carbamyl phosphate synthetase I and argininosuccinate synthetase. mRNAs for these two enzymes exhibit distinctive patterns of expression in cultured rat hepatocytes. Recombinant DNA constructs incorporating genomic DNA sequences for these enzymes and a reporter gene will be used in cell transfection experiments to identify and characterize cis-acting DNA sequences involved in the response to hormones. Preliminary experiments will also be undertaken to identify DNA-binding proteins with sequence- specific affinity for regions of DNA shown by transfection experiments to be important for expression.

对当前生物学中心的主要兴趣问题不仅在 激素调节的机制问题 特定基因的表达，但也以坐标的方式表达 基因的表达 - 例如，对于在A内的单个酶的表达 代谢途径 - 已实现。 基因的表达 编码尿素周期的酶构成了极好的 用于研究此问题的两个方面的模型。 尿素 循环由2个线粒体和3种胞质酶组成 由核基因编码 仅在尿路动物的肝脏中很大。 哺乳动物中尿素周期酶的水平对 饮食蛋白质含量，糖皮质激素，胰高血糖素的变化， 胰岛素和甲状腺激素。 该实验室的结果 就像其他人一样，已经证明了主要组成部分 这些变化涉及改变的细胞浓度 这些酶的mRNA。 该实验室的研究也 证明这些基因在大鼠中的转录率 肝脏因环状AMP而增加，并且 糖皮质激素和二丁扬酰环状AMP都是必要的，并且 足以提高大鼠尿素周期酶的mRNA水平 在无血清培养基中培养的肝细胞。 该赠款提出孤立和表征基因组DNA 编码两个尿素周期酶的克隆 - 卡木基 磷酸合成酶I和精氨酸合成酶。 mrnas 对于这两种酶，表现出独特的表达方式 在培养的大鼠肝细胞中。 重组DNA构建体 将这些酶和A的基因组DNA序列纳入 报告基因将用于细胞转染实验 识别和表征与涉及的顺式作用DNA序列 对激素的反应。 初步实验也将是 承诺鉴定具有序列的DNA结合蛋白 转染显示的DNA区域的特异性亲和力 实验对于表达很重要。