TRANSKINGDOM CONJUGATION OFFERS A POWERFUL GENE TARGETING TOOL IN YEAST

Gene targeting is one of the powerful techniques used to investigate e ukaryotic genes. In a typical eukaryotic microbe, Saccharomyces cerevi siae yeast, we examined trans-kingdom conjugation between Escherichia coli bacterium and yeast as a gene targeting tool. Here, it is shown t hat trans-kingdom conjugation effectively induced gene replacement eve n on yeast's target loci (e.g. ura3-52 allele) which is never targeted by conventional transformation. This clearly indicates that trans-kin gdom conjugation offers a very powerful gene targeting tool in yeasts. In fact, Southern hybridization analysis of transconjugants distinctl y verified the accuracy in the conjugative gene replacement. The effic iency of gene replacement was about 0.4 x 10(-7) per recipient yeast. This is enough to sustain gene targeting with gene replacement by tran s-kingdom conjugation. We also discuss the mechanism of conjugative ge ne replacement. (C) 1998 Elsevier Science B.V.