Genetic selection of meiotic and mitotic recombinant yeast artificial chromosomes

We have developed a genetic screen for the isolation of larger or smaller recombinant yeast artificial chromosomes derived from overlapping YACs. Integration plasmids were used to modify the TRP1 and URA3 auxotrophic markers present respectively on the left and right vector arms of one of the parental YACs. Diploids containing the two parental YACs were studied through meiosis and mitosis. Tetrad analysis revealed the presence of meiotic recombinant YACs at a frequency comparable with what is expected for yeast DNA (about 3 kb/cM). More direct genetic selection of diploids on -TRP-LYS synthetic media in the presence of 5-fluoro-orotic acid (5-FOA), led to the isolation of mitotic recombinant YACs at a high frequency. Analysis of these yeast cells by pulsed-field gel electrophoresis, confirmed the loss of both parental artificial chromosomes, and the specific retention of a larger or smaller recombinant YAC.