engleski

Genetic side effects accompanying gene targeting in yeast: the influence of short heterologous termini

We investigated the influence of short terminal heterologies on recombination between transforming linear DNA fragments and the yeast Saccharomyces cerevisiae genome. The efficiency of plasmid integration to the CYC1 locus (ends-in assay) was decreased more than five-fold when the size of terminal heterology exceeded 28 nucleotides (nt) and a similar inhibitory effect was also observed in the ends-out assay (replacement of the ura3-52 allele by the UPIA3 gene). Plasmid integration occurred almost exclusively in the target homology and was accompanied by excessive degradation of the heterollogous termini. Illegitimate integrations were much more frequent in the ends-out transformation in both the absence (8.9%) and the presence (23.7%) of 45/46 heterologous nucleotides at the ends of the transforming fragment. Interestingly, only about 60% of transformants arose by simple gene replacement, regardless of the presence of heterologous ends, whereas more complex interactions resulted in gene or whole chromosome duplications. Our results warn that different genetic alterations may be introduced in the host strain during ends-out transformation but also indicate possible mechanisms for formation of duplications in the genome.

gene targeting; gene duplication; aneuploidy; homologous recombination; illegitimate recombination

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