Functional analysis of imprinted gene Peg3.

印迹基因Peg3的功能分析。

• 批准号: 13680909
• 负责人: KOHDA Takashi
• 金额: $ 2.62万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680909/
• 关键词: 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

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.

小鼠Peg 3基因是一个只在父本等位基因上表达的印迹基因。Peg 3基因编码C2 H2型锌指结构域蛋白，在小鼠哺乳行为、肿瘤进展和细胞凋亡中具有重要作用，为了阐明Peg 3的生物学功能，我们利用酵母双杂交系统筛选了Peg 3结合蛋白。以GAL 4融合蛋白Peg 3为诱饵筛选小鼠脑cDNA，从4 × 16^6个cDNA克隆中分离到10个候选基因。将这些候选cDNA与Flag-tag融合，并与Myc标记的Peg 3在HeLa细胞中表达。其中一个候选物通过免疫沉淀和蛋白质印迹法在体内与Peg 3结合。该cDNA克隆编码近全长的过继蛋白14-3-3 eta的编码区。重新检查的氨基酸序列的Peg 3，我们发现一个共识模体的14-3-3结合在N-末端部分的Peg 3蛋白。由于该蛋白在Hela细胞中的不相容性，我们目前还不能在体内证实其它候选cDNA克隆的Peg 3结合活性。