Construction of versatile Escherichia coli--yeast shuttle vectors for promoter testing in Saccharomyces cerevisiae.

The promoterless PHO5 gene of the yeast Saccharomyces cerevisiae, encoding the repressible acid phosphatase (AP) was utilized as a reporter gene for the construction of a novel vector system for selection and functional analysis of yeast promoters. The Escherichia coli-yeast shuttle plasmids, pZHB81 and pZHB82, contain different arrays of unique restriction sites located upstream of the PHO5 coding region. Yeast promoters could be screened from random DNA fragments (cloned in the upstream sites) for their ability to direct the expression of the PHO5 gene in transformed (AP-deficient) yeast host cells. AP-expressing transformants were selected directly on agar plates by using a routine colony staining method. Relative promoter strength was assessed by direct assay for AP activity in cell lysates.