engleski

Genetic changes introduced by illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

Integration of the exogenous DNA into the yeast genome is achieved by homologous recombination ; in the absence of homology between plasmid DNA and yeast chromosome only rare integration events can be observed. We have found that the efficiency of such illegitimate integration can be enhanced up to 100 fold if single-stranded circular (ssc) or linearized (ssl) plasmids are used for transformation. Transformants were analyzed by Southern blotting of their genomic DNA that was either cut with different restriction enzymes or resolved in individual chromosomes by pulse-field gel electrophoresis. Our results indicate that some chromosomes are preferred targets for illegitimate integrations, but the presence of the vector sequence was also detected in non-chromosomal DNA. Ethidium bromide stained gels revealed chromosomal size polymorphism among transformants which could not be explained only by multiple plasmid integrations. Transformation with ss plasmid linearized in the region with homology in the yeast genome also resulted in illegitimate integrations. However, several observations indicate that the ssl DNA can also be involved in homologous interactions with the yeast chromosome. We present a model for illegitimate integration of ss DNA based on these observations.

yeast; single-stranded DNA; illegitimate recombination

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