engleski

Transformational replacement of heterology in the yeast genome

Efficient homologous recombination in the yeast Saccharomyces cerevisiae makes possible the use of simple procedures for the disruption of target genes. One of the methods available is based on transformational replacement of the chromosomal sequence with linear DNA fragment engineered in vitro. Typically, the coding region of the cloned gene is replaced by another gene that may be used for selection after which this construct, together with flanking regions of the target gene, is used for transformation. Recombination between free ends of the transforming DNA and homology on the chromosome results in substitution of the chromosomal sequence with linear fragment containing disrupted gene. This method is successfully used for gene disruption/replacement within the entire yeast genome but is also studied as a model system for gene "knock out" in other organisms. Here we report the results of our experiments where we performed the reverse reaction in which the interrupted sequence was replaced by the wild type gene in order to recover functional gene product. We also asked if this simple technique can be used to replace large insertions in the chromosome with another insertion present on the linear fragment in the one-step insertion replacement reaction. Two in vitro engineered linear DNA fragments were used to transform yeast strain containing the ura3-52 allele, in which the URA3 gene is disrupted by Ty1 insertion (6.1 kb). The first transforming fragment contained the intact yeast URA3 gene (1.17 kb) while the second one contained the yeast ARG4 gene (2.06 kb) inserted approximately in the middle of the URA3 gene (ura3::ARG4 construct). Accordingly, the Ura+ transformants were expected to arise by conversion of the Ty1 insertion and the Arg+ transformants by replacement of the genomic Ty1 insertion with the exogenous ARG4 insertion. This was confirmed by Southern blotting analysis on enzymatically digested genomic DNA of Ura+ and Arg+ transformants. However, for the Arg+ transformants, the dominant genetic event was not the replacement of insertion, but the addition of transforming DNA into the homology present in the yeast genome. Our results have shown that the transformational replacement of large heterology in the yeast genome can be achieved in one-step reaction. We also propose a molecular model for the non-conservative addition in which transforming DNA gets integrated into disrupted homology on the yeast chromosome.

gene replacement; Saccharomyces cerevisiae; genetic recombination

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