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[Study on a downstream signal molecule of human CASK/LIN-2].

OBJECTIVE
To elucidate the function of human CASK/LIN-2, a novel member of membrane-associated guanylate kinase homologs family (MAGUK), by using the yeast two-hybrid system(LexA) to screen the protein interacting with the guanylate kinase-like domain(GK) of hCASK and identify possible protein that might involve downstream signal transduction of hCASK.


METHODS
PCR strategy was used to amplify GK domain cDNA fragment of hCASK. The DNA was subcloned into pLexA to construct bait vector pLexA-GK. The pLexA-GK was transformed into yeast host strain EGY48; through testing the bait protein and a repression assay, it was demonstrated that the bait protein did not have transcription activation for leu and lacZ reporter genes, however it could enter nucleus, bind LexA operator. Human fetus brain cDNA library plasmids were transformed into EGY48 containing pLexA-GK and p8op-lacZ, and screened on plates of Gal/Raf his- trp- ura- leu- and Gal/Raf his- trp- ura-X-gal. The clones with galactose dependent leu+ and lacZ+ were isolated; their library plasmids were rescued by means of transforming E.coli KC8. These library plasmids were transformed into the yeast again to re-screen. The remaining positive clones were analyzed by PCR and restriction endonuclease. Finally two kinds of target DNA fragments were obtained.


RESULTS
The specificity testing indicated that the two target proteins could specially bind GK domain of hCASK. After DNA sequencing and BLASTn analysis on NCBI, it was shown that the two target DNA fragments, 732 and 683 bp, had high sequence similarity with human inhibitors of differentiation 1(Id1) mRNA, with identities of 97%(630/645) and 98%(656/666), respectively.


CONCLUSION
GK domain of hCASK could specially interact with Id1 in yeast. Id1 might be a downstream signal molecule of hCASK, which might involve in regulation of cell differentiation.