METABOLIC EFFECTS OF THYROID HORMONE IN CELL CULTURE

利用培养的GH1和GC细胞，两种生长激素产生大鼠垂体 L-三碘甲腺原氨酸(L-T_3)对血管内皮细胞有刺激作用。 由刺激引起的生长激素mRNA积累的增加 生长激素基因转录。在这些细胞中，糖皮质激素 激素也与L-T3协同作用，刺激转录 生长激素基因。这项提议的一个目标是绘制DNA控制图 生长激素基因的激素调节元件 表情。我们构建了一个嵌合基因(pGH-xgpt)，该基因由 大鼠生长激素基因5‘侧翼区1.8kb， 连接到含有结构基因的细菌DNA上，该结构基因编码 黄嘌呤-鸟嘌呤磷酸核糖转移酶(XGPT)。在……里面 SGPT的PGH-xgpt表达受任何激素调控 生长激素基因5‘端的元件。这一构造 获得稳定的GC细胞转化子，频率较高 其中XGPT基因受L-T3的高度调控。5‘端缺失文库 将构建突变体以定位 通过激素调节表达的DNA元件。DNA是否 生长激素基因转录起始点的3‘序列 还将探索是否可以调节表达。地区 利息将被细分，并使用稳定的AS进行详细的进一步分析 以及瞬间的表情。我们还将评估5‘侧翼是否 人生长激素基因区域在GC中受激素的调节 细胞来评估激素控制元件的可能同源性 物种线。其他研究将与荷尔蒙的位置有关 大鼠生长过程中核酸酶敏感部位的调控元件 荷尔蒙。不同的方法将探索 具有确定的生长激素基因片段的甲状腺激素受体。vbl.使用 L-T_3(L-T_3-PAL)的光亲和标记衍生物 分子量(MR)的甲状腺激素受体形式：47,000和56,000。 将使用光亲和标记来阐明 这些磁共振形式及其与基因片段和染色质的相互作用 域名。光亲和标记也将被用来识别可能的 患者成纤维细胞甲状腺激素受体的微妙变化 有甲状腺激素抵抗的人。

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.