REGULATION OF HEPATOCELLULAR FUNCTION BY GROWTH HORMONE

本申请中提出的实验旨在研究 生长激素的作用机制。为了实现这一目标，增长 激素反应的肝脏信使RNA序列将被定义，并 生长激素和其他因子对这些mRNA序列的协调调节 荷尔蒙将会被测定。此外，关于是否会有 生长激素在特定的mRNA序列上引起的变化是 增加的转录和/或稳定的mRNA序列将 接受检查。将使用两种方法来测量组织水平 特定的mRNAs序列。作为生长发育的初步筛选技术 激素反应的mRNA序列，总mRNA将从肝脏中分离出来 来自体内处理的动物或来自培养的肝细胞。信使核糖核酸将 用于指导无细胞兔网织红细胞的蛋白质合成 同位素标记氨基存在下的裂解产物翻译系统 酸和标记的蛋白质将用二维凝胶分离 电泳法。单个光斑强度，它反映了 将通过视频评估该斑点的功能mRNA编码量 成像。为了确认体外翻译系统的结果，并 为了进一步研究感兴趣的信使核糖核酸序列，互补DNA探针 将为生长激素反应序列做准备。重组 将以含肝聚(A)的RNA为起点制备质粒 互补DNA插入物的材料。改造后的殖民地将是 用Poly(A)RNA制备的[32P]cDNA进行菌落杂交筛选 从甲状腺切除或生长激素治疗中获得 切除甲状腺的动物。来自产生的菌落的质粒DNA 差分信号将用于混合选择的翻译和 体外合成产物经凝胶电泳法鉴定。 生长激素反应基因序列的合适的cDNA探针 将用于测量肝细胞中该信使核糖核酸的合成速率 培养和从对照和生长激素处理获得的细胞核中 组织。通过类似的技术，特定信使核糖核酸的降解速度 还将接受检查。这些研究将为以下方面提供重要的见解 生长激素在翻译前水平的作用机制和 为实验奠定了基础，旨在研究 激素-质膜受体相互作用和翻译前事件。 此外，对人类生长发育障碍的全部复杂性的洞察 将会有所收获。

The experiments proposed in this application are designed to investigate the mechanism of growth hormone action. To achieve this objective, growth hormone responsive hepatic messenger RNA sequences will be defined and the coordinate regulation of these mRNA sequences by growth hormone and other hormones will be determined. In addition, the question of whether the changes induced by growth hormone on specific mRNA sequences are the result of increased transcription and/or stabilization of the mRNA sequence will be examined. Two approaches will be used to measure tissue levels of specific mRNA sequences. As an initial screening technique for growth hormone responsive mRNA sequences, total mRNA will be isolated from livers of in vivo treated animals or from cultured hepatocytes. The mRNA will be used to direct protein synthesis in the cell free rabbit reticulocyte lysate translational system in the presence of isotopically labeled amino acids and the labeled proteins will be separated by two-dimensional gel electrophoresis. Individual spot intensity which is a reflection of the quantity of functional mRNA coding for that spot will be assessed by video imaging. To confirm the results of the in vitro translational system and to further study the mRNA sequences of interest, complementary DNA probes will be made for the growth hormone responsive sequences. Recombinant plasmids will be prepared using hepatic poly(A)-containing RNA as starting material for the complementary DNA inserts. Transformed colonies will be screened by colony hybridization with [32P] cDNA prepared from poly(A) +RNA obtained either from thyroidectomized or growth hormone treated thyroidectomized animals. Plasmid DNA from colonies which yield differential signals will be used for hybrid selected translation and the in vitro synthesized product identified by gel electrophoresis. Appropriate cDNA probes to the growth hormone responsive mRNA sequences will be used to measure rate of synthesis of that mRNA in hepatocyte cultures and in nuclei obtained from control and growth hormone treated tissue. By similar techniques the rate of degradation of specific mRNA will also be examined. These studies will provide significant insight on the mechanism of growth hormone action at a pretranslational level and provide the foundation for experiments designed to examine the link between hormone-plasma membrane receptor interaction and pretranslational events. In addition, insight into the full complexity of disorders of growth in man will be gained.