I propose to investigate the functions encoded by the yeast plasmid, 2mu circle, and determine the mechanism by which expression of these functions is achieved. One approach is analogous to a strict genetic analysis. I will introduce small deletions in vitro into coding regions of 2mu circle species, into a nonessential region of which has been inserted in the LEU2 gene of yeast to allow selection of the mutated plasmid in yeast. These mutated plasmids will be introduced into circle plus or circle minus yeast strains, by transformation. The resulting transformants will be examined to determine whether the plasmid has lost the ability to enhance transformation, replicate, or catalyze intramolecular recombination, and, if so, whether this loss of function can be complemented by genes present on endogenous 2mu circle. A complementary approach to this will be to transform circle plus and circle minus yeast strains with hybrid pBR322-LEU2 plasmids containing various restricting fragments of 2mu circle to determine whether the presence of the 2mu circle sequences will promote enhanced transformation by and/or replication of the hybrid plasmid in yeast. This approach will also allow the localization and definition of the 2mu circle origin of replication. In addition, I propose to develop several assays for 2mu circle encoded proteins to permit their purification. In this way the enzymatic activities of these proteins can be correlated with their functions as determined above. In a second series of experiments, I will examine transcription of 2mu circle to determine whether, as our previous data has suggested, 2mu circle specific messenger RNAs are derived by processing larger precursor transcripts and whether expression of specific messenger RNA is determined by the intramolecular recombination observed in 2mu circle. Specifically, these experiments involve introducing transcriptional steps into various regions of the molecule and examining the pattern of transcripts derived from the resultant plasmids. Thus, these experiments will serve to broaden our understanding of mechanisms of gene expression in eucaryotes. In addition, these results will promote the analysis of eucaryotic DNA replication by enhancing the value of 2mu circle as a model system.
