Construction of chimeric genes for elusidating unique properties of amylase-III

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From cultured-cells of rice (Oryzae sativa cv Sasanishiki), we previously isolated and characterized two a-amylase isozymes (designated as AMY-I and AMY-III). One of them, AMY-III,showed a low temperature optimum of 25ﾟC unlike that for other plant a-amylases including AMY-I,thet generally had around 50ﾟC.To know what is responsible for these unique properties of the AMY-III,we isolated and characterized cDNA clones for AMY-I and-III,and studied the expression of those and the chimeric genes with various combinations of AMY-I cDNA and AMY-III cDNA in Saccharomyces cerevisiae.Secondary structure analysis of AMY-III according to the method of Chou and Fasman revealed that the region-c lying between the concerved regions B and C showed b-sheet structure, on the other hand, AMY-I and other well studied amylases, namely A.oryzae amylase (TAA), porcine pancreatic amylase (PPA) have shown a-helix structutre for this region In fact, chimera protiens obtained from the yeasts carrying a plasmid of pGMlu III I or pGSca III I or pGSal I III,all of which contain region-c of AMY-I showed characteristic properties of AMY-I,and these were all secreted proteins. On the contrary, other type of chimera proteins obtained from the yeasts carrying a plasmid of pGNae III I or pGMlu I III or pGSca I III,all of which contain region-c of AMY-III,were not secreted in the medium and immunoblotting analysis showed the same mobility to AMY-III that prepared from yeast cells carrying a vector, pG-3 with AMY-III.This indicates AMY-III and AMY-I-AMY-III chimera proteins carrying region-c of AMY-III accumulate intracellularly in yeast. These results suggest that the region-c of AMY-III protein determines the characteristic properties of AMY-III.