engleski

Analysing targeted chromosome duplication in yeast Saccharomyces cerevisiae by qPCR

Gene targeting is one of the basic tools in genetic engineering allowing deletion, precise modification or insertion of genes thus facilitating new strain construction for research and industry. Gene targeting is based on transformation of cells with a linear DNA fragment carrying ends homologous to the targeted region. The desired result of such transformation is replacement of the targeted region of the genome with the transforming DNA fragment. Yeast Saccharomyces cerevisiae is known for extraordinarily efficient gene targeting, i.e. the occurrence of unwanted, aberrant genetic events during gene targeting in this organism is generally considered to be fairly low. The most frequent aberrant genetic event in yeast is the targeted chromosome duplication, where one chromosome copy carries the transforming DNA in the targeted region while the other chromosome copy retains the original allele. In recent study we used qPCR to analyse the targeted chromosome duplication events in more detail. We used telomere-proximal primer pairs and probes to confirm that indeed the entire length of the targeted chromosome is duplicated. Moreover, we determined not only the targeted chromosome copy number, but also the copy number of all other (non-targeted) chromosomes. Interestingly, only two out of eight analysed transformants were aneuploids carrying targeted chromosome duplication and a single copy of non-targeted chromosomes, while other six out of eight transformants were diploids. Based on this results, we proposed a two-step mechanism for genome duplication instigated by gene targeting. After the duplication of the targeted chromosome (by break-induced replication mechanism), a protein content imbalance occurs. Since the transformants were kept under selective pressure to maintain both targeted chromosome copies, the imbalance could not be relieved by loss of an extra chromosome copy. Therefore, the duplication of non-targeted chromosomes leading to diploidisation and restoration of protein content balance would be favoured in yeast cell culture.

gene targeting, targeted chromsome duplication, genome duplication

nije evidentirano