Selection Of The Proteins Interacting With AA12 And CHK1

A1-5 and B4 were derived from primary rat embryo fibroblast cell lines, as compared with its counterparts B4, A1-5 shows extreme radioresistance to killing accompanied by an unusually strong G2 checkpoint responses. PCR-Select cDNA Subtractive hybridization was used to screen the differential expression genes of this two cell lines. AA12 gene was obtained by using this method; it was found that AA12 gene was high expression in A1-5 cells and low in B4 cells.AA12 and Chk1 genes were inserted into the pGBKT7 and pGADT7 vector of two-hybrid system, and the recombinant plasmids were transformed into yeast cell AH109. The HIS3 and ADE2 report genes were identified on SD/-Ade/-His/-Leu/-Trp agar plates, and lacZ and MEL1 genes were identified by α- and β-Galactosidase Assays. The result showed that AA12 and CHK1 could interact with each other in yeast. But we cannot make sure whether AA12 gene plays an important role to an unusually phenotypes of the A1-5 cells.To screen new proteins that can interact with AA12 or CHK1 and confirm the interaction between them, AA12 and Chk1 genes were inserted into the pGBKT7 vector of two-hybrid system respectively, and the recombinant plasmids were transformed into yeast cell AH109 first. The expression of AA12 was identified by Western blot. Then the prostate cDNA library recombinant plasmids were transformed into the yeast cells. When AA12 was used as bait protein, 30 positive clones were obtained with α- and β-Galactosidase assays, further matting assay showed only two of them were positive. The sequencing and BLAST indicated that both of them were homologous with ribosomal protein L41 (RPL41). When using CHK1 as a bait protein, 102 positive clones were obtained, 4 of them were positive in matting assay only three chromosome genomic contigs sequences were identified. The interaction between target proteins and screened proteins still need to be confirmed and the mechanism needs to be studied further.