Strategy for modifying and duplicating target chromosomes. (A) PCR amplifies PGAL1-CEN3 URA3 from the plasmid pGALCEN-JC3-13  and, upon transformation into yeast, replaces the target centromere by homologous recombination. (B) The HIS3 plasmid pKA52 is integrated into URA3 adjacent to the conditional centromere, disrupting the URA3 open reading frame and generating direct repeats (shaded). At an approximate frequency of 10-4, HIS3 is lost by homologous recombination between the direct repeats, regenerating a functional URA3 gene (reverse arrow). The recipient strain carries the deletion alleles ura3 Δ0 and his3-Δ200. (C) A haploid carrying a modified chromosome from (B) is grown in galactose for one cell division, generating N+1 and N-1 cells by nondisjunction. Since the ura3::HIS3 marker is present in two copies, cells with URA3 and HIS3 can be produced by HIS3 excision and are identified as Ura+His+ papillae on selective medium.