Inducible expression of REP1 causes inducible expression of the 2 micron circle stability system

The yeast plasmid, 2 micron circle, encodes a stability system consisting of the plasmid replication origin, a cis-active locus designated REP3 and two trans-active functions--the products of the REP1 and REP2 genes. We have constructed 2 micron circle derivatives in which the expression of the REP1 gene is placed under the control of the yeast GAL10 promoter. We show that in such plasmids the stability-system is inducible, being turned off by glucose and turned on by galactose. Further, our results unequivocally demonstrate that, of the two potential in-frame ATG codons at which REP1 translation might initiate (as inferred from the 2 micron circle DNA sequence and from the cap site of the major REP1 transcript), the upstream ATG is dispensable without affecting REP1 function. We also illustrate here a simple and general method for constructing in vivo in yeast 2 micron circle analogs which contain desired alterations within specific regions of the 2 micron circle genome.