Molecular cloning of thyroid hormone receptor interacting protein using yeast two hybrid system
| Project/Area Number |
07671126
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
内分泌・代謝学
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| Research Institution | Nagoya University |
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Principal Investigator |
NAGAYA Takashi Nagoya Univ.Res.Inst.Environ.Med.Research Associate, 環境医学研究所, 助手 (80262913)
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| Co-Investigator(Kenkyū-buntansha) |
KAMBE Fukushi Nagoya Univ.Res.Inst.Environ.Med.Research Associate, 環境医学研究所, 助手 (00211871)
MURATA Yoshiharu Nagoya Univ.Res.Inst.Environ.Med.Associate Professor, 環境医学研究所, 助教授 (80174308)
SEO Hisao Nagoya Univ.Res.Inst.Environ.Med.Professor, 環境医学研究所, 教授 (40135380)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Thyroid hormone receptor / TR interacting protein / Yeast two hybrid system / Co-repressor / Thyroid hormone / Cloning / Two hybrid system / 分子生物学 |
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| Research Abstract |
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 PROTEIN
Molecular 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
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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 LINES
Gene 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
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