Fig. 15. Two-step gene replacement. This procedure allows the experimenter to introduce any type of mutation anywhere in the yeast genome, without leaving behind vector sequences or a selectable marker. The desired mutation with flanking sequences is cloned into a conventional plasmid that also contains a marker that can be selected both for and against. URA3 and ura4+ are usually employed for this purpose in S. cerevisiae and S. pombe, respectively. Then the plasmid is digested with a restriction enzyme that cuts once within the yeast sequences flanking the mutation and is introduced into yeast by transformation. The DNA break stimulates recombination within the flanking sequences, leading to integration of all the vector sequences and the mutated yeast sequences into the chromosome of the recipient yeast cell. Note that there is a duplication of the target sequences, with one copy containing the mutation and the other copy retaining the wild type sequence. The successfully transformed cells are selected by growth in medium lacking uracil. Some of these cells are then subjected to counter selection in medium containing uracil and 5'-fluoroorotic acid (FOA), which kills cells capable of uracil biosynthesis. Under these conditions, only cells which have "popped out" the vector sequences and the URA3 or ura4+ marker by recombination between homologous duplicated sequences (the region containing a, b, c and d or a', b', c' and d') can survive and grow. If the recombination occurs in the c, d region to the right of the mutation, the mutation is popped out and the cell becomes wild type again. If the recombination is in the a, b region to the left of the mutation, then the wild type sequence is popped out and the cell becomes mutant.