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 URA3 gene). Plasmid integration occurred almost exclusively in the target homology and was accompanied by excessive degradation of the heterologous 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.
Svetec
Ivan-Kresimir
IK
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia.
Stafa
Anamarija
A
Zgaga
Zoran
Z
D001483:Base Sequence; D045304:Cytochromes c / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*
C014059:CYC1 protein, S cerevisiae; D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c