| Project/Area Number |
13680909
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Laboratory animal science
|
|---|
| Research Institution | Tokyo Institute of Technology |
|---|
Principal Investigator |
KOHDA Takashi Tokyo Institute of Technology, Gene Research Center, Assistant Professor, 遺伝子実験施設, 助手 (60211893)
|
|---|
| Project Period (FY) |
2001 – 2002
|
| Project Status |
Completed (Fiscal Year 2002)
|
| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
|
|---|
| Keywords | Peg3 / 14-3-3 / two-hybrid system / protein binding / ゲノミックインプリンティング / zinc finger protein / 蛋白質相互作用 / インプリンティング / two hybrid / 母性行動 |
|---|
| Research Abstract |
Mouse Peg3 is an imprinted gene exclusively express from paternal allele. This gene encodes C2H2 type zinc finger motif containing protein and known to have important roles in nursing behavior in mice, tumor progression in and cell apoptosis.
To elucidate the biochemical function of Peg3, we have screened the Peg3 binding proteins using yeast two-hybrid system. Through the screening of mouse brain cDNAs by GAL4 fused Peg3 as bait, 10 candidate gene were isolated from 4x16^6 cDNA clones. These candidate cDNAs were fused with Flag-tag and expressed with Myc-tagged Peg3 in HeLa cells. One of the candidates was proved to bind in vivo with Peg3 by immuno-precipitation followed by western blotting. The cDNA clone encodes near full-length coding region of adopter protein 14-3-3 eta.
Re-examining the amino acids sequence of the Peg3, we found a consensus motif for 14-3-3 binding in the N-terminal part of Peg3 protein. It is also proved that the binding activity was destroyed when the serine residue within the motif changed to alanine by site directed mutagenesis.
Until now, we couldn't proof the Peg3 binding activity of other candidate cDNA clones in vivo because of the insulubility of the protein in Hela cells.
|
