Molecular cloning of thyroid hormone receptor interacting protein using yeast two hybrid system

甲状腺激素对正常生长和发育以及几种代谢途径至关重要。这种作用是通过其核受体介导的，核受体主要与9-顺式维甲酸受体（RXR_s）以异二聚体形式结合于DNA序列。DNA上的这种复合物可以以配体依赖的方式激活或抑制甲状腺激素应答基因。最近的研究表明，TR β可以与基础转录机制的组成部分相互作用，这些相互作用是转录调控的关键。此外，TRs和基础转录机制之间的中介辅因子被推测对转导激素信号很重要。甲状腺激素受体相互作用蛋白的克隆利用酵母双杂交系统对甲状腺激素受体相互作用蛋白（辅因子）进行了分子克隆。以人甲状腺激素受体为筛选载体，构建了含Ga 14激活结构域的Hela细胞cDNA文库 关于我们 在Ga 4 DNA结合域载体（pGBT 9）中的orbeta（TRbeta）。分离出一个与TRbeta相互作用的克隆（命名为B1），并通过核苷酸测序认为其是小鼠核受体辅阻遏物（N-CoR）的人类同源物。利用噬菌斑杂交法对Hela cDNA文库进行筛选，获得了该克隆的全长，并从中分离到与克隆B1同源性为80%的克隆H3。氨基酸（a.a.）克隆H3推导出的氨基酸序列在其羧基末端不同，克隆B1的120 aa被不同的50 aa取代。由于mN-CoR通过其羧基端区域与TR和视黄酸受体（RAR）相互作用，因此N-CoR变体（克隆H3）羧基端的改变可能影响受体相互作用的特异性。N-CoR异构体的存在将支持这些异构体的不同表达可能调节甲状腺激素的转录调节的可能性。用RT-PCR方法分析N-CoR亚型在细胞中的基因表达。从10个不同的细胞系中提取RNA进行逆转录，然后用PCR扩增N-CoR异构体，在所有研究的细胞系中，检测到两种N-CoR异构体的mRNA表达。这表明N-CoR亚型广泛表达。少

Thyroid hormone is essential for normal growth and development as well as several metabolic pathways. This effect is mediated through its nuclear receptors, which bind to the DNA sequences mainly as a heterodimer with 9-cis retinoic acid receptors (RXR_s). This complex on DNA can activate or repress thyroid hormone responsive genes in a ligand dependent manner. Recent studies indicated that TR complexe can interact with components of basal transcriptional machinery and that these interaction is critical for transcriptional regulation. Furthemore, the intermediating co-factors between TRs and basal transcriptional machinery are speculated to be important for transducing hormonal signals.1. CLONING OF THYROID HORMONE RECEPTOR INTERACTING PROTEINMolecular cloning of thyroid hormone receptor interacting proteins (co-factors) was performed using yeast two hybrid system. Hela cell cDNA library constructed in Ga14 activation domain vector (pGAD-GH) was screened by human thyroid hormone recept … More orbeta (TRbeta) in Ga4 DNA binding domain vector (pGBT9). One clone (named B1)to interact with TRbeta was isolated and considered to be a human homologue of mouse nuclear receptor co-repressor (N-CoR) by nucleotide sequencing. As Hela cDNA library was screened by plaque hybridization method to obtain full length of this clone, another clone (named H3) which is 80% homology with clone B1 was isolated. The amino acids (a.a.) deduced by clone H3 were different in its carboxy-terminus, in that 120a.a.of clone B1 was replaced with distinct 50a.a.. Since mN-CoR interacts with TR and retinoic acid receptor (RAR) through its carboxy-terminal region, the alteration of carboxy-terminus in N-CoR variant (clone H3) might affect specificity of receptor interactions. The existence of N-CoR isoforms will support the possibility that the different expression of these isoforms may modulate transcriptional regulation by thyroid hormone.2. EXPRESSION OF N-COR ISOFORMS IN THE CELL LINESGene expression of N-CoR isoforms in the cells was analyzed by RT-PCR method. The extracted RNA from 10 different cell lines was reverse-transcribed and followed by the amplification of N-CoR isoforms with PCR.In all cell lines studied, mRNA expression of two N-CoR isoforms were detected. It indicates that N-CoR isoforms are expressed ubiquitously. Less