Formation of Extrachromosomal DNA Rings in Saccharomyces cerevisiae Using Site-Specific Recombination

A hindrance to the study of structure and function of DNA elements is that sites of interest always lie within the context of other DNA sequences. This is particularly limiting when attempting to examine elements embedded within chromosomes inside intact cells. Analysis of the Saccharomyces cerevisiae genome has shown that genes are densely packed and dispersed among multiple replication origins, as well as other functional loci. The importance of context is exemplified by the phenomenon of transcriptional silencing, where regions of inactive chromatin repress the expression of proximal genes. A solution to the problem of context has been to relocate elements of interest to naturally occurring or synthetic plasmids. Though small in size and simple in organization, biologically sustainable plasmids are still complex. Shuttle vectors used in both yeast and bacteria must contain sequences necessary for replication and selection in both hosts. Many shuttle vectors also contain an additional DNA element that determines whether the plasmid will be maintained at high or low copy.