THYROID HORMONE RECEPTOR AND GENE EXPRESSION

甲状腺激素受体和基因表达

• 批准号: 3240058
• 负责人: HOWARD C TOWLE
• 金额: $ 8.93万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240058
• 关键词: DNA binding protein; antireceptor antibody; complementary DNA; gene expression; genetic library; genetic manipulation; genetic mapping; genetic transcription; hormone receptor; laboratory rabbit; laboratory rat; molecular cloning; protein engineering; protein sequence; protooncogene; receptor expression; transfection; triiodothyronine

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.

这项研究的远距离目标是探索分子 核甲状腺激素受体介导的基础 特定基因表达的变化。 最近的报告有 建议甲状腺激素受体是正常的 V-erba癌基因的细胞对应物。 我们将测试这个 通过分离大鼠肝脏的全长cDNA来假设 与V-erba癌基因同源。 抗体的能力 针对纯化的肝C-ERBA多肽饲养，产生 在大肠杆菌中，以识别核甲状腺激素结合 将测试大鼠肝脏的活性。 此外，肝C-Erba 适当的胞源表达载体中的cDNA将是 引入缺乏甲状腺激素的肝癌细胞 受体。 核甲状腺激素结合的表达 甲状腺激素反应基因的活性和调节 将测量转染后。 最后，能力 表达的C-ERBA多肽与特定DNA结合 甲状腺激素反应性基因（S14基因）的序列 并将评估成绩单。 初步证据表明，至少有两个Erba- 在大鼠中表达的同源基因。 我们建议 替代的erba基因可以编码变体形式 甲状腺激素受体的靶基因不同 特殊性。 我们进一步建议这样的特异性 替代形式的受体将由70氨基确定 涉及与DNA结合的酸性结构域。 为了检验这一假设， 将选择与ERBA相关cDNA克隆的替代形式 从适当的大鼠cDNA文库（例如，大脑）。 序列 这些变体形式将与成年肝脏中发现的形式进行比较， 特别是在DNA结合域中。 表达 不同组织和发育中的变体C-ERBA基因 将测试大鼠的阶段。 C-变体形式的能力 ERBA多肽与肝的结合并刺激表达 将确定甲状腺激素反应基因。 最后， 使用体外诱变的技术， C-ERBA变体形式的DNA结合域将是 系统地更改为更像肝 形式。 这些改变形式的c-erba与 S14基因上的DNA序列，并刺激 该基因将被监测。 这些研究应该提供见识 进入在氨基酸残基中起着至关重要的作用 确定与DNA靶的相互作用的特异性 站点。