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Targeted chromosome duplication as a side-efect of gene targeting in yeast

Gene targeting by introduction of linear, non-replicative DNA fragments relies on cell's DNA repair mechanisms to replace the genomic allele with the transforming one. Apart from routine use for the construction of different strains, this procedure is also studied as a general model for targeted genetic manipulations by the ends-out recombination. It is generally assumed that, in contrast to higher eukaryotes, transformation of the yeast Saccharomyces cerevisiae with non-replicative DNA occurs almost exclusively by homologous recombination. We performed a systematic study of gene replacement using several URA3 alleles present both on the transforming DNA fragment and in the yeast genome. Depending on the experimental system, targeted gene replacement ranged from 98 % to only 60 %. Molecular analysis of transformants revealed versatile aberrant recombination events including illegitimate integration of transforming DNA, gene or whole chromosome duplications and chromosome fusions suggesting that the fidelity of gene targeting in yeast is mainly influenced by the structure of alleles involved in recombination. We propose that ends-out recombination involving endogenous DNA fragments, released from two close double-strand-breaks, may play an important role in the duplication of chromosomal fragments or whole chromosomes.

genetic duplications; gene targeting; ends-out recombination; gene replacement; yeast Saccharomyces cerevisiae

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