Genetic screening of the yeast gene disruption library using an efficient plasmid transfer method.
Rodney Rothstein, Ivana Sunjevaric, Robert J.D. Reid
Genetics & Development, Columbia University Med. Ctr., 701 W 168th Street, New York, NY, 10032-2704, USA
We developed an efficient method to transfer plasmid DNA into any ura3-Saccharomyces strain. This mating-based method uses a kar1 plasmid donor strain that initiates mating but cannot form a diploid and can allow plasmid transfer (plasmoduction) between nuclei in the heterokaryon. To overcome the requirement for specialized genetic backgrounds in the recipient strains as well as spurious chromosome transfer, we constructed a universal donor strain containing a conditional centromere and a URA3+ allele on all 16 chromosomes. Plasmoduction with this strain only requires that the recipient be ura3, GAL+ and contain another marker for selection of the transferred plasmid. Counter selection against every donor chromosome also limits spurious allele transfer. We have screened the gene disruption library for mutations sensitive to overexpression (synthetic dosage lethality) of either the TOP1 gene or a dominant negative TOP1 allele (T722A) that traps topoisomerase I with DNA similar to camptothecin. We found 11 mutations unable to tolerate Top1 overexpression including one that was also identified as synthetic lethal in the absence of TOP1. The screen with Top1-T 722 A identified over 100 mutants including many that were previously identified in camptothecin screens. Among the new mutations, we found 9 of the 12 genes involved in the maturation of the pre-vesicle compartment into multi-vesicular bodies linking this step in vesicular movement to DNA/topoisomerase metabolism.