METABOLIC EFFECTS OF THYROID HORMONE IN CELL CULTURE

用培养的GH-1和GC细胞， 细胞系，我们已经表明，L-三碘甲状腺原氨酸（L-T3）刺激， 刺激导致生长激素mRNA积累增加 生长激素基因的转录。 在这些细胞中， 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 生长激素基因的激素调节元件 表情 我们已经构建了嵌合基因（pGH-xgpt），其由以下组成： 1.8大鼠生长激素基因的5 '侧翼区， 连接到含有编码以下结构基因的细菌DNA上 黄嘌呤-鸟嘌呤磷酸核糖转移酶（XGPT）。 在 SGPT的pGH-xgpt表达受任何激素控制的调节 在生长激素基因的5'区域中的元件。 该构建体 产生GC细胞的稳定转化体，其相对高的频率在 其中XGPT基因受L-T3高度调控。 5'缺失文库 将构建突变体以定位所述细胞的位置和边界。 通过激素介导调控表达的DNA元件。无论是DNA 生长激素基因转录起始位点的3'序列 也将探索如何调节表达。 地区 兴趣将被细分，并使用稳定AS 以及瞬时表达。 我们还将评估5 '侧翼是否 人生长激素基因调控区在胃癌中的作用 细胞，以评估激素控制元件的可能同源性， 物种线 其他的研究将把激素的位置 调控元件对大鼠生长的核酸酶超敏感位点 荷尔蒙Goene。 各种方法将探索 具有确定的生长激素基因片段的甲状腺激素受体。 使用 L-T3的光亲和标记衍生物（L-T3-PAL），我们已经鉴定了两种 分子量（Mr）甲状腺激素受体形式; 47，000和56，000。 光亲和标记将用于阐明 这些MR形式及其与基因片段和染色质的相互作用 域. 光亲和标记也将用于鉴定可能的 甲状腺成纤维细胞甲状腺激素受体的细微变化 甲状腺激素抵抗

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.