engleski

Integration of plasmid DNA with short heterologous ends in Saccharomyces cerevisiae

We studied the influence of short terminal heterology on plasmid integration in the genome of the yeast Saccharomyces cerevisiae. Yeast cells were transformed with nonreplicative plasmid linearized within the CYC1 region containing short (104 bp) heterologous insertion. Different restriction enzymes were used to introduce double-strand break (DSB) or deletion within the heterology, or just in the junction between the CYC1 gene and insertion. The presence of heterology at both ends of the linearized plasmid resulted in 20-fold decrease in the efficiency of transformation while the presence of one heterologous end caused only two to four-fold decrease. We used plasmids with identical or different heterologous ends and they gave basically the same results. Southern blot analysis of genomic DNA was used to further characterise the nature of transformation events. 97/100 transformants were produced by plasmid integration in the yeast CYC1 region and only three by illegitimate integration somewhere else in the yeast genome. This result shows that the presence of short heterologous ends has no significant influence on targeting of plasmid integration. However, in 19/97 transformants obtained by homologous integration plasmid-derived heterology was detected in the CYC1 region suggesting that repair of DSB by homologous recombination can occur without elimination of entire terminal heterology from the invading DNA molecule.

DSB repair; plasmid integration; yeast

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