METABOLIC EFFECTS OF THYROID HORMONE IN CELL CULTURE

使用培养的GH 1细胞，生长激素产生大鼠垂体细胞系，我们 已经表明，L-三碘甲状腺原氨酸（T3）刺激增加的速度， 生长激素合成和mRNA积累。 这种反应似乎是 由可从细胞核中提取的染色质相关受体介导 通过0.4 M KCl和蔗糖梯度中的沉积物作为3.8 S物种。 在 相比之下，微球菌核酸酶消化切除受体作为一个主要的6.5 S 12.5S的形式，其沉积速度略快 比产生的大部分单核体（11.5S）。 在完整细胞T3中 引起受体水平的时间依赖性降低， 依赖于配体-受体相互作用。 此外，核水平 Bounds受体与染色质乙酰化程度呈负相关 相关蛋白质 我们已经开发了密度标记方法来定量 核受体的合成和降解，我们打算检查细胞 控制稳态核受体水平的机制。 我们还打算 以确定受体是否以细胞质形式存在， 作为核结合形式的前体。 我们合成了一个亲和标记 其与受体结合位点共价连接。 我们会用这个作为辅助 在受体纯化和研究受体的一级结构方面。 利用这些受体制剂，我们建议制备单克隆抗体 使用杂交瘤技术用于进一步的受体纯化和作为 研究染色质中受体相互作用的额外工具。 我们将 检查6.5S和12.5S的形成过程中的代谢产物关系 受体物种，并确定它们是否来自 染色质的转录活性区域。 无论受体是 与染色质中的其他蛋白质相关的蛋白质将通过化学方法进行评估。 交叉研究。 最后，利用基因克隆技术，我们将研究 T3增加生长激素mRNA积累的机制， GH 1细胞。

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.