THYROID HORMONE RECEPTOR AND GENE EXPRESSION

这项研究的长期目标是探索分子 核甲状腺激素受体介导的基础 特定基因表达的变化。最近的报道说 提示甲状腺激素受体是正常的 V-erba癌基因的细胞对应物。我们将对此进行测试 通过分离大鼠肝脏的全长cDNA提出假说 与v-erba癌基因同源。抗体的能力 抗纯化的肝脏c-erba多肽，生产 在大肠杆菌中，识别核甲状腺激素结合 将测试大鼠肝脏的活性。此外，肝脏c-erba 合适的真核表达载体中的cDNA会被 导入缺乏甲状腺激素的肝癌细胞 受体。核甲状腺激素结合蛋白的表达 甲状腺激素应答基因的活性及其调控 随后的转染率将被测量。最后，有能力 表达的c-erba多肽与特异性DNA的结合 甲状腺激素应答基因(S14基因)的序列分析 它的成绩单将被评估。 初步证据表明，至少有两个额尔巴人- 在大鼠体内表达的同源基因。我们建议 替代的erba基因可以编码不同形式的 靶基因不同的甲状腺激素受体 具体细节。我们进一步提出，这种情况的特殊性 受体的替代形式将由70个氨基酸决定 与DNA结合有关的酸性结构域。为了检验这一假设， 将选择不同形式的Erba相关的cdna克隆 从适当的大鼠c DNA文库(例如，脑)。按顺序排列 这些变异形式将与成人肝脏中发现的进行比较， 尤其是在DNA结合域中。的表达方式 C-erba基因在不同组织和发育中的变异 将对不同阶段的老鼠进行测试。C-变种形式的能力 Erba多肽与肝脏结合并刺激其表达 甲状腺激素反应基因将被确定。最后， 利用体外诱变技术， 不同形式的c-erba的DNA结合域将是 系统地改变以更接近于肝脏 形式。这些改变形式的c-erba结合到 S14基因上的DNA序列及对其表达的刺激作用 这一基因将受到监测。这些研究应该会提供洞察力 转化为氨基酸残基，这些残基在 确定与DNA靶标相互作用的特异性 网站。

The long range goal of this research is to explore the molecular basis by which the nuclear thyroid hormone receptor mediates changes in specific gene expression. Recent reports have suggested that the thyroid hormone receptor is the normal cellular counterpart of the v-erbA oncogene. We will test this hypothesis by isolating full-length cDNA for rat liver which are homologous to the v-erbA oncogene. The ability of antibodies raised against the purified hepatic c-erbA polypeptide, produced in E. coli, to recognize the nuclear thyroid hormone binding activity in rat liver will be tested. In addition, the hepatic c-erbA cDNA in an appropriate eucaryotic expression vector will be introduced into hepatoma cells lacking the thyroid hormone receptor. The expression of nuclear thyroid hormone binding activity and regulation of thyroid hormone responsive genes following transfection will be measured. Finally, the ability of the expressed c-erbA polypeptide to bind to specific DNA sequences of a thyroid hormone responsive gene (the S14 gene) and it transcript will be assessed. Preliminary evidence suggest that there are at least two erbA- homologous genes which are expressed in the rat. We propose that alternate erbA genes could encode variant forms of the thyroid hormone receptor which differ in their target gene specificities. We further propose that the specificity of such alternate forms of receptor will be determined by the 70 amino acid domain involved in binding to DNA. To test this hypothesis, alternate forms of erbA-related cDNA clones will be selected from appropriate rat cDNA libraries (eg., brain). The sequence of these variant forms will be compared to that found in adult liver, particularly within the DNA-binding domain. The expression of variant c-erbA genes in different tissues and developmental stages of the rat will be tested. The ability of variant forms of c- erbA polypeptide to bind to and stimulate expression of hepatic thyroid hormone responsive genes will be determined. Finally, using the techniques of in vitro mutagenesis, sequences within the DNA-binding domains of variant forms of c-erbA will be systematically changed to more closely resemble the hepatic form. The ability of these altered forms of c-erbA to bind to DNA sequences on the S14 gene and to stimulate the expression of this gene will be monitored. These studies should provide insight into the amino acid residues which play a critical role in determining the specificity of the interaction with DNA target sites.