Trans-kingdom conjugation offers a powerful gene targeting

Abstract

Gene targeting is one of the powerful techniques used to investigate eukaryotic genes. In a typical eukaryotic microbe, Saccharomyces cerevisiae yeast, we examined trans-kingdom conjugation between Escherichia coli bacterium and yeast as a gene targeting tool. Here, it is shown that trans-kingdom conjugation effectively induced gene replacement even on yeast's target loci (e.g. ura3-52 allele) which is never targeted by conventional transformation. This clearly indicates that trans-kingdom conjugation offers a very powerful gene targeting tool in yeasts. In fact, Southern hybridization analysis of transconjugants distinctly verified the accuracy in the conjugative gene replacement. The efficiency of gene replacement was about 0.4×10⁻⁷ per recipient yeast. This is enough to sustain gene targeting with gene replacement by trans-kingdom conjugation. We also discuss the mechanism of conjugative gene replacement.