Efficient expression of theEscherichia coli leuB gene in yeast

Summary

Efficient expression of theEscherichia coli ZeuB (ß-isopropylmalate dehydrogenase) gene occured in yeast after in vitro DNase digestion and religation of plasmid bound ZeuB and the yeastIIIS3 DNA which placed the 5′ end of the yeastHIS3 gene immediately adjacent to the coding region of theE. coli leuB gene. Two structurally distinct classes of gene fusions were constructed, each involved portions of the yeastHIS3 gene which contributed DNA sequences responsible forleuB expression in yeast. The first class involved fusion of theHIS3 coding region to bacterial DNA resulting in the production of a fusion protein with ß-isopropylmalate dehydrogenase activity. The second class consisted of bacterial DNA, including theleuB coding region, fused to theHIS3 promotor region with the absence of any portion of theIIIS3 coding region. In both constructions theIIIS3 promotor region is required for transcription, however, translation of the class two fusion is initiated at a bacterial DNA coded AUG, and the 5′ end of the mRNA coded by theleuB gene mapped predominantly at bacterial DNA sequences. The DNA sequence responsible for the 5′ end of theHIS3 mRNAs remain in the class two gene fusions but this did not preclude the initiation of transcription at bacterial DNA sequences. The pattern of mRNA initiation at bacterial DNA suggests that DNA sequences at, or adjacent to, the site of transcription initiation are involved in the determination of the sites of initiation, and perhaps the frequency at which initiation occurs.