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. 2005 Jul 25;33(13):4128–4139. doi: 10.1093/nar/gki727

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© The Author 2005. Published by Oxford University Press. All rights reserved

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Construction of tsn⁻ and tsn⁻trax⁻ mutants. S.pombe tsn⁻ strains were generated by first deleting one of the two Translin alleles in a diploid. This was accomplished by replacement of one tsn allele with a ura4 gene, through homologous recombination with a linear DNA fragment containing the ura4 open reading frame flanked on both sides by sequences that also flank the tsn gene. Next, meiosis was induced and a tetrad analysis was performed. Haploid tsn⁻ colonies were found in the tetrads. We obtained a trax⁻ S.pombe strain, which was generated by Decottignies et al. (23), and crossed this strain with a tsn⁻ haploid. Meiosis was induced again and strains, in which both Translin and TRAX have been deleted, were found among the haploid descendents of the diploids generated by the cross. (A) Scheme of the heterozygous diploid, tsn⁺/tsn⁻. Primers P1 and P2 were used for PCR analysis to confirm the indicated gene replacement. The expected lengths of the PCR products before and after the gene replacement are indicated. (B) Tetrad analysis of the heterozygous diploid strain tsn⁺/tsn⁻. Four viable spores were obtained in all tetrads. The larger colonies were shown to be ura4⁺,tsn⁻. (C) PCR analysis of a tsn⁺haploid (lane 1), a heterozygous diploid tsn⁺/tsn⁻ (lane 2), a haploid tsn⁻::ura4 (lane 3) and a haploid tsn⁻::ura4, trax⁻::kanR (lane 4). The expected length for a tsn⁺ product (1.8 kb) and a tsn⁻ product (2.8 kb) are shown by a closed and an open arrowhead, respectively. (D) Scheme for trax gene replacement. The TRAX open reading frame has been replaced in S.pombe by the kanamycin-resistant gene (kanR) (23). We used the primer P3 and P4 for PCR analysis designed to confirm the gene replacement. The expected lengths of the PCR product before and after the gene replacement are indicated. (E) PCR analysis of a haploid strain trax⁺ (lane 1), a haploid strain trax⁻::kanR (lane 2) and a haploid strain tsn⁻::ura4, trax⁻::kanR (lane 3). The expected length for a trax⁺ product (1.4 kb) and a trax⁻ product (2.1 kb) are indicated by closed and open arrowheads, respectively.