1998 Fiscal Year Final Research Report Summary
Functional analysis of thyroid hormone receptor interacting protein
| Project/Area Number |
09671045
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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 Res.Inst.Environ.Med., Nagoya Univ., Research Associate, 環境医学研究所, 助手 (80262913)
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| Co-Investigator(Kenkyū-buntansha) |
KAMBE Fukushi Res.Inst.Environ.Med., Nagoya Univ., Associate Prodessor, 環境医学研究所, 助教授 (00211871)
MURATA Yoshiharu Res.Inst.Environ.Med., Nagoya Univ., Professor, 環境医学研究所, 教授 (80174308)
SEO Hisao Res.Inst.Environ.Med., Nagoya Univ., Professor, 環境医学研究所, 教授 (40135380)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Thyroid hormone receptor / TR interacting protein / Yeast two hybrid system / Co-repressor / Thyroid hormone / Cloning |
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| Research Abstract |
To understand the molecular aspect of thyroid hormone action, we performed to clone TR interacting protein by yeast two hybrid system. Hela cell cDNA library constructed in Gal4 activation domain vector (pGAD-GH) was screened by human thyroid hormone receptor beta (TRbeta) in Gal4 DNA binding domain vector (pGBT9). One done (named B1) to interact with TRbeta was isolated and considered to be a partial fragment of human nuclear receptor co-repressor (hN-CoR) by nucleotide sequencing. By colony hybridization technique and 5'-RACE, we cloned wild type and other three variants of hN-CoRs. One variant (hN-CoRv1) is 56 amino acids (a.a.) insertion in to a.a. 1018 of wild type N-CoR.The second one (hN-CoRv2) is a deletion of 120 a.a between a.a. 1857 and 1977. The third one is a carboxyterminal variant, in that 120 a.a. of wild type sequence is replaced with distinct 50 a.a..
To study the chromosomal localization of hN-CoR, fluorescence in situ hybridization technique was performed using hN-Co
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R cDNA as a probe. Then the locus of hN-CoR was assigned to chromosome 17p11.2. Since hN-CoR localizes at one locus, the variants are considered to be generated by alternative splicings.
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.
Expression profile of N-CoR variants was analyzed in 8 human cell lines by RT-PCR method. The extracted RNA was reverse-transcribed and followed by the amplification of N-CoR variants with PCR.In all cell lines studied, mRNA expression of two N-CoR variants (hN-CoRv2 and v3) were detected minimally in comparison with Wild type. However, the expression of hN-CoRv1 was different in the cell lines. In three cell lines (NB-1 neuroblastoma cell line, Hela cervival carcinoma cell line and HOS osteosarcoma cell line), hN-CoRv1 was expressed predominant than wild type. The diffrent expression of hN-CoR variants might modify repression level by co-repressor and modulate hormonal signalings. Less
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