Current Studies of Biotechnology – Volume II. - Environment PLASMID INTEGRATION IN YEAST: CONCEPTIONS AND MISSCONCEPTIONS ZORAN ZGAGA ∗ , KREŠIMIR GJURAČIĆ 1 , IVAN-KREŠIMIR SVETEC, PETAR T. MITRIKESKI AND SANDRA GREGORIĆ Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; *PLIVA Research institute,Zagreb, Croatia Abstract Integration of exogenous DNA into the yeast genome occurs by homologous recombination and is both directed and stimulated by the presence of double-strand breaks (DSBs) in transforming DNA. Repair of the break with or without plasmid integration occurs with equal probability and it is generally assumed that no information is lost or gained during this process. However, some important assumptions of current models are challenged by the results obtained by our group. First, we showed that rare illegitimate integrations may also occur in the presence of homology in the yeast genome and that integration in homology does not always result in reconstruction of the functional allele. Second, the presence of point mutations in double-stranded, but not in single-stranded plasmid, decreased integration in homology. However this antirecombinogenic effect was not proportional to the number of mutations. Third, targeted integrations were strongly suppressed by the presence of short heterologous sequences at the ends of the linearized plasmid. Terminal heterology is not always eliminated during integration and this process can lead to the generation of new alleles. In our experimental system, less then 10% of transformants that repaired DSB present on the replicative plasmid contained the plasmid sequence in their chromosomal DNA. We propose that the plasmid integration can be stimulated by repair processes operating on the hybrid DNA formed during recombination. Key words: gene targeting, genetic recombination, Saccharomyces cerevisiae Introduction Application of different technologies for gene transfer and/or modification is often limited by recombinational machinery of the host cell. Illegitimate integration of exogenous DNA is associated with alterations in the organization of genetic material that may lead to unpredictable changes in cell physiology and genetic stability. Unfortunately, this seems to be the prevailing mechanism of genetic transformation in a number of organisms, including human cells. Theoretically, this problem could be circumvented either by stimulation of homologous recombination, or by suppression of illegitimate integration and much effort has been made in order to better understand these processes. For the study of homologous recombination, the yeast Saccharomyces cerevisiae is still the model organism of choice. Although illegitimate events have also been described and characterised in yeast (1, 2, 3) homologous integration occurs with high frequency and is routinely used for manipulation of yeast genes. In first transformation experiments circular, non-replicative plasmids were found to recombine with homology present in the genomegiving rise to plasmid integration or to genetic conversion of the mutated allele (4). Later it was shown that restriction enzyme-induced double-strand breaks (DSBs) on the plasmid molecule stimulate transformation and direct recombination process to the homology present in the yeast genome (5). These experiments were the basis for development of the DSB model for genetic recombination (6), but also presented the first example of gene targeting technology. Subsequently, another procedure based on homologous recombination was developed, making possible gene replacement/disruption in a single step (7). By different experimental strategies yeast genes can now be mutated, deleted, replaced or cloned and foreign DNA can be introduced into any desired location within the genome. ∗ Corresponding author address: Zoran Zgaga, Faculty of Food Technology and Biotechnology, Pierotti St. 6, 10000 Zagreb, Croatia