| Popis: |
It is generally assumed that, in contrast to higher eukaryotes, gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, we found that even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out (gene replacement) and ends-in (plasmid integration) transformation assays. It was determined that ends-out gene targeting can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, ends-in gene targeting is often associated with multiple targeted integration events but illegitimate integration is extremely rare while, so far targeted chromosome duplication has not been reported. Here we systematically investigated the influence of the design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. We have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1 sgs1 double mutant suggesting that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting. |
