STRATEGY FOR DELETION OF COMPLETE OPEN READING FRAMES IN SACCHAROMYCES-CEREVISIAE

The classical disruption method for yeast genes is by using in vitro d eletion of the gene of interest, or of a part of it, with restriction enzymes. We are now routinely using a strategy that takes advantage of polymerase chain reactions (PCRs) which amplify large pieces of DNA. Since this approach results in a complete, precise deletion of the ope n reading frame, which is replaced by a unique restriction site, the l igated PCR can be used for the insertion of different markers or for t wo-step gene disruptions without an inserted marker. As we have now us ed this strategy for the deletion of more than ten genes we have in th is report included some hints based on our experience.