engleski

Yeast transformation and horisontal gene transfer

In contrast to the vertical inheritance of genetic traits, from parents to their offspring, the acquisition of genetic material from neighbors may involve members of different species. The incoming genetic material has first to find a way to enter the recipient cell and then to become established there. Two examples of such horisontal gene transfer acquired much attention due to their great practical importance: plasmid shuttling between bacterial species and unidirectional transfer of genetic information from the procaryote Agrobacterium tumefaciens to the plant cells where the single-stranded DNA from bacterial origin integrates in the plant genome by illegitimate recombination. The exogenous DNA, coming from another species, is expected to have no homology, or only limited homology to the host genome. How could such heterologous DNA integrate into the genome of the yeast Saccharomyces cerevisiae, whose recombinational machinery is known to operate efficiently only with homologous DNA molecules? We developed several experimental systems, based on yeast transformation, to determine homology requirements for integration of foreign DNA in the yeast genome. First, we determined the proportion of illegitimate events for the plasmid cut within the region homologous to the yeast genome. As expected, more than 99,5% of transformation events were due to the integration in homology. Addition of short heterologous sequences at the ends of linearized plasmid decreased the efficiency of transformation, but did not increase the yield of illegitimate integrations. We also studied the effects of low (0.1-1%) and moderate (8,6%) sequence divergency on plasmid integration as well as integration of plasmids with no homology in the yeast genome. Our results can be summarized as follows: (i) recombination between non identical (homeologous) duplexes was decreased, but not completly abolished, while heterologous plasmids integrated with extremly low efficiency and (ii) heterologous DNA in single-stranded form was found to be highly (up to hundred-fold) more proficient in illegitimate integration. These results suggest that yeast can accomodate foreign double-stranded DNA only from closely related species, while the DNA in single-stranded form, like that transferred in conjugation with bacteria, could bridge large evolutionary barriers.

genetic transformation; gene transfer; single-stranded DNA; Saccharomyces cerevisiae

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