Functional analysis of imprinted gene Peg3.

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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.