Fig. 14. One-step gene replacement in yeast. Within a cloned stretch of yeast chromosomal DNA containing two restriction enzyme sites (EndoR1 and EndoR2), a marker gene selectable in yeast is inserted by standard cloning procedures. Additional alterations, such as deletion of yeast sequences and/or point mutation of yeast sequences can also be carried out. All such alterations should be >60 bp from the restriction sites, so that at least 60 bp of homologous sequence flanks the mutated region on both sides. Then the mutated segment of yeast chromosomal DNA is excised from the vector by restriction digestion at the two sites, and it is introduced into yeast cells by standard transformation procedures. The ends of the restriction fragment are recombinogenic, and the desired double recombinants can be selected by growth on medium requiring the selectable marker. When the selectable marker and/or mutation are expected to destroy the function of a gene, it is best to carry out this procedure in diploids. Otherwise, if the destroyed gene is essential, no cells containing the desired mutation will survive.