| Project/Area Number |
16590902
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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 |
Endocrinology
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| Research Institution | Nagoya University |
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Principal Investigator |
KANOU Yasuhiko Nagoya Univ., Research Institute of Environmental Medicine, Assistant professor, 環境医学研究所, 助手 (50252292)
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| Co-Investigator(Kenkyū-buntansha) |
MURATA Yoshiharu Nagoya University, Research Institute of Environmental Medicine, Professor, 環境医学研究所, 教授 (80174308)
TAKAGISHI Yoshiko Nagoya University, Research Institute of Environmental Medicine, Assistant professor, 環境医学研究所, 助手 (50024659)
SEO Hisao Nagoya University, Research Institute of Environmental Medicine, Professor, 環境医学研究所, 教授 (40135380)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Resistance for thyroid hormone / thyroid hormone receptors / coactivators / Yeast two-hybrid system / cDNA / Cloning |
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| Research Abstract |
We tried to identified novel proteins which can specifically bind to thyroid hormone receptor (TR) b using yeast two-hybrid system.
{Method}
Yeast two-hybrid Matchmaker System 3 (Clontech, Palo Alto, CA, USA) was used to screen for novel TR-interacting proteins. A human TR b full length cDNA was cloned into pGBKT7 plasmid vector which contained GAL4 DNA binding domain. We screened a human liver cDNA library (Clontech) using the human TR cDNA as bait in the yeast two hybrid system.
{Results}
The mating efficiency and number of clones screened corresponded to the values indicated in the manufacture's instructions.
1) In the 1st screening we got 131 clones on the plates supplemented with T3 (+T3). The additional screening was yeast clones were streaked both on the +T3 and -T3 plates. Ninety one clones grew in the T3 dependent manner. The plasmids of these yeast clones were purified and the inserts were sequenced. Two unknown sequences were identified ; No. 60 and 93.
2) The full length of 2 clones were cloned from human liver total RNA by RT-PCR. Three different mRNAs of the clone No. 60 and a mRNA of No.93 were found in the liver. To determine whether TR physically interact with either the products of clone 60 or 93, the plasmid for GST fusion proteins were constructed.
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