Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates

Abstract

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.

FIG. 1

Physical structure of the RDN1 locus of strain MRY247. The bars represent the tandemly repeated units of the locus. The solid bars represent units of rDNA. The open bars represent insertions of plasmid pAA11, whose genetic map is shown at the top. The shaded bars represent insertions of plasmid pMR4, whose genetic map is shown at the bottom.

FIG. 2

Physical maps of pMR4, pAA11, and plasmids recovered from E. coli clones transformed by DNA of S. cerevisiae MRY247. (A and B) Plasmids recovered from clones transformed by untreated DNA. (C through G) Plasmids recovered from clones transformed by DNA treated with HindIII and ligase. Only restriction sites relevant for characterization of the plasmids are shown. Open and solid arrowheads indicate restriction sites that are critical for identification (XbaI in pMR4 and SmaI in pAA11, respectively). The solid bars in maps B, E, F, and G represent DNA regions that were not characterized.