New vectors for construction of recombinant high-copy-number yeast acentric-ring plasmids.

Yeast acentric-ring plasmid 1 (YARp1), comprising 1453 bp of entirely yeast chromosomal DNA, is maintained in Saccharomyces cerevisiae as a high-copy, relatively stable plasmid. To determine the feasibility of using YARp1 as a yeast cloning vehicle, we subcloned the GAL1-10 promoter and the URA3 gene into YARp1 at different locations. To facilitate these constructions, a class of permuted YARp1 construction vectors was generated which enabled us to use various restriction sites in YARp1 as insertion points. Transformation frequencies, plasmid stabilities, and copy numbers of these YARp1 derivatives remained elevated, comparable to those of YARp1 itself. Also, when OMP decarboxylase was assayed using strains containing URA3-YARp's, specific activities of 100-300 times that of wild type were found. This evidence supports the use of YARp1 as a high-copy yeast-expression vector or for analyzing structural and regulatory DNA sequences.