Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene EN01

Abstract

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene ( EN01) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of EN01 were isolated from this EN01- lacZ fusion plasmid by in vitro recombination. Analysis of β-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the EN01 - lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of EN01. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.