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

Abstract

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.

Different Aneuploidies Arise From the Same Bridge-Induced Chromosomal Translocation Event in Saccharomyces cerevisiae

“…Another explanation for these phenomena could be that, in adapted cells that are able to bypass cell cycle arrest, the recombination of the BIT cassette interferes with chromosome segregation during mitosis, giving rise to the aneuploidy noted for both chromosomes XVI and IX. Missegregation due to interchromosomal association as described (Kaye et al 2004) was also proposed to be linked to the generation of disomic cells following the integration of a DNA construct with short heterologous termini in S. cerevisiae (Svetec et al 2007). A defect in chromosome segregation, possibly due to the size differences of chromosome IX and the newly formed aberrant chromosome, might account for the duplication or triplication of chromosome IX observed in the SUSU4 and SUSU8 strains.…”

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

“…A high fidelity of gene targeting (percentage of a successful gene targeting) has also been reported for the moss Physcomitrella patens [27, 28]. Moreover, when flanking homologies are not of the same length, the transforming DNA fragment integrates next to the sequence sharing longer homology, frequently creating tandem repeats as previously reported in the moss [29], mouse cell lines [30] and yeast [14]. …”

“…Plasmid pAGUS was constructed by replacing PvuII-PvuII fragment (2038 bp) containing 1638 bp URA3 region of the plasmid pAGU2 with the SmaI-SmaI fragment (1104 bp) carrying shorter URA3 region from the plasmid pTZGU [14]. Standard media and procedures were used for the cultivation of the E. coli strains (DH5α and XL1blue) and DNA manipulations [49].…”

“…In EIGT, the double-strand break (DSB) on the linearized plasmid is almost always repaired by HR resulting in targeted plasmid integration, whereas the frequency of random (non-targeted) integration due to illegitimate recombination could be estimated to be as low as 0.1 % [14]. Moreover, illegitimate integration, including homology-assisted illegitimate recombination [15, 16] is the only aberrant event observed so far.…”

“…Contrary to EIGT, the spectrum of aberrant events during EOGT is wider. Apart from (i) random integration by illegitimate recombination; transformation can result in (ii) integration of the transforming DNA fragment next to the homologous target, generating tandem repeats and (iii) targeted chromosome duplication (TCD) detected in aberrant transformants containing both, allele expected after a successful gene targeting and an untransformed allele (heteroallelic transformants) [14]. Duplication of a targeted chromosome can occur either by extensive DNA synthesis from the 3′-ends of the transforming fragment to the ends of the targeted chromosome by break-induced replication (BIR) [17, 18] or by chromosome missegregation [19].…”