CONSTRUCTION OF MULTICOPY YEAST PLASMIDS WITH REGULATED CENTROMERE FUNCTION

The promoter region from the cloned glucose-repressible alcohol dehydogenase II (ADH2) gene has been inserted in front of the centromere III (CEN3) sequence. It has been shown that, in plasmids containing ADH2-CEN3 fusion (YCRp plasmids), the CEN3 function can be regulated by glucose repression. Wen yeast transformed with YCRp plasmid are grown in the presence of glucose, the ADH2 promoter is repressed and CEN3 functions normally, mitotically stabilizing the YCRp plasmid. When the carbon source in the culture medium is substituted with ethanol, the transcription from the derepressed ADH2 promoter interferes with the CEN3 function, rendering the YCRp plasmids unstable. At that stage YCRp2 plasmids may accumulate to about ten copies per cell whereas YCRp3 copy number increases to about 100. This demonstrates that the CEN3 sequence can be present on high copy number plasmids in yeast with no detectable toxic effects.