Mitotic Gene Conversion, Reciprocal Recombination and Gene Replacement at the BenA, Beta-tubulin, Locus of Aspergillus Nidulans

We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5'-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomyl-resistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6 x 10(-4). Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0 x 10(-4) and gene conversion leading to benomyl-resistant conidia occurred at a rate of 2.6 x 10(-4). We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.