Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance

Abstract

Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.