The yeast plasmid, 2 micron circle, and hybrid plasmids derived from it are more stable than most plasmids of similar size constructed from chromosomal ARS sequences. Previous studies in this and other laboratories have suggested that this can be attributed to a plasmid encoded system for maintenance of a high, regulated copy number and for equipartitioning of plasmids in mitotic and meiotic cell divisions. Our current efforts are focused on identifying the plasmid components responsible for these systems and elucidating their regulatory interactions. We have shown that equipartitioning of plasmid molecules requires a cis-acting sequence, REP3, and two plasmid genes, REP1 and REP2. We have purified the REP1 protein and shown that it is a component of the yeast nuclear matrix. Further, we have found that it undergoes calcium induced aggregation and binds specifically to discrete plasmid DNA sequences. We have also raised antibodies to the REP2 protein and identified it in extracts of plasmid-bearing yeast cells. We plan to continue these studies by precisely defining the nature of the interaction between the components of this system - namely, REP1 protein, REP2 protein, specific 2 micron sequences, and the nuclear matrix - and by delineating in more detail the characteristics of the partitioning process. The FLP protein of 2 micron circle catalyzes site-specific recombination between two repeated segments present on the plasmid. We have shown that this system enables the plasmid to increase its mean intracellular copy number in yeast cells growing under non-selective conditions by a FLP-induced transient shift in the mode of replication from theta to double rolling circle. We are pursuing this system by identifying intermediates in amplification. In addition, we are developing it as a system to promote directed plasmid and chromosomal rearrangements in yeast for use in addressing various novel questions of recombination and cell structure. Evidence exists to indicate that 2 micron circle controls its copy level through autogenous regulation of expression of its various products. We are using beta-galactosidase gene fusions to the four 2 micron circle genes in conjunction with constructs consisting of promoter fusions to the 2 micron circle coding regions to provide a complete description of 2 micron circle's regulatory circuitry.
