Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region

Abstract

Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA). Transformation can be achieved either by using purified plasmid DNA or by fusion of yeast spheroplasts with partially lysed Escherichia coli [pLC544] protoplast preparations. The Trp+ yeast transformants are highly unstable, segregating Trp- cells at frequencies of 0.18 per cell per generation (haploids) and 0.056 per cell per generation (diploids) in media containing tryptophan. Plasmid pLC544 replicates autonomously in the nucleus of yeast cells and segregation of Trp-cells is associated with the complete loss of plasmid sequences. In genetic crosses, pLC544 is randomly assorted during meiosis and is carried unchanged through the mating process into haploid recombinants.