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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Genetic reporter for recombination between heteroalles residing on homologous chromosomes in tetraploid strains.
(A) The tetraploid cells carry two versions of chromosome II with deletions within the TYR1 ORF: the “ty”-allele contains a 5′ portion of the TYR1 ORF (nucleotides 1–700) and the “yr1”-allele contains a 3′ portion (nucleotides 300–1358). The green box represents the ORF of the TYR1 gene; the striped boxes represent the missing DNA sequences of the mutants; and the light blue rectangle represents the area of homology between the chromosomes. Note: for simplicity only one of each pair of sister chromatids of the G2/M cells are presented (thereby, having the appearance of G1 cells). (For a complete description of recombination and segregation at mitosis see Figure S7.) Gene conversion with or without crossing-over as well as reciprocal exchange between heteroalleles can generate Tyr+ cells. Recombination leading to Tyr+ can generate different combinations of TYR1 alleles within the resulting tetraploid cell. A large conversion tract will result in 3 types of alleles regardless of associated crossing-over: the original truncated parental heteroalleles and the TYR1+ converted allele. A reciprocal exchange that occurs at short conversion region will yield a forth allele “y” retains only a small portion of the gene. Depending on segregation of sister chromatids at mitosis, half the Tyr+ recombinants that arise by a reciprocal exchange would not possess the “y” allele (for details see Figure S7). (B) The 4 alleles described in (A) can be distinguished by size of PCR products using the following primers: 5′GAATACCGTAGCACTTGAAGGAAAGAGGACAGCATATCCA5′CACAAAAGAAGGCCTAATATTATAGGAAATCAGCATTAAAAAC. The allele sizes are 1360 bp (TYR1), 1060 bp (“yr1”), 700 bp (“ty”) and 400 bp (“y”). Presented are PCR products of the TYR1 locus from 4 colonies obtained after UV irradiation (40 J/m2) of the MCD1 simplex strains. Tyr+ colonies that result from a reciprocal exchange event can be identified by the presence of a “y” allele (encircled). The “M” corresponds to DNA molecular size markers.