| dc.description | The 2mum circle is an endogenous plasmid stably maintained in the nucleus of the budding yeast Saccharomyces cerevisiae. The aim of this research has been to gain an understanding of the maintenance of the 2mum circle, with particular focus on the 2mum-encoded Rep proteins Rep1 and Rep2 and a cis-acting site, STB, involved in mediating 2mum circle segregation. To analyze 2mum plasmid segregation, a 2mum-based plasmid (pAS4) which includes all the 2mum sequences required for segregation, was constructed. In addition, it carries the yeast ADE2 gene and sequences that allow the plasmid to be propagated and selected in E. coli. Data show that this plasmid segregates as efficiently as the native 2mum, despite lacking the FLP gene required for plasmid amplification. pAS4 is the first artificial plasmid that is as stable as native 2mum circle. The presence of the ADE2 gene has allowed segregation of this plasmid to be studied in an ade2 mutant yeast host strain, using a pink/white colour assay system. The assay system was used to screen for mutant alleles of REP1 and REP2 generated by polymerase chain reaction (PCR) and introduced into the pAS4 plasmid by gap repair. pAS4 was also used to assess the ability of full-length and truncated REP genes to provide segregation function. A two-hybrid analysis was used to define the domains of interaction of the Rep proteins. The results indicated that the amino-terminal domains of the two Rep proteins are sufficient for their interaction, while in vivo complementation showed that both the amino and carboxy terminal regions of the Rep proteins are required for segregation function. The data from these studies have been used to develop a model that predicts how the interaction between the Rep proteins and their association with the cis-acting STB locus results in segregation of the 2mum circle. | en_US |
