On homologous recombination in Aspergillus nidulans.

This thesis deals with the molecular mechanisms that underlie homologous recombination in eukaryotes. Most molecular genetic studies on homologous recombination have been performed in budding yeast ( Saccharomyces cerevisiae ). However, it remains to be investigated to what extent the results obtained in this unicellular organism can be extrapolated to other eukaryotes. We therefore initiated a series of molecular genetic studies on homologous recombination in Aspergillus nidulans . This filamentous fungus is amenable to most molecular techniques and is phylogenetically closer to higher eukaryotes than S. cerevisiae ; A. nidulans may thus provide a powerful eukaryotic model system. We have examined meiotic and mitotic recombination events in the nii A- nia D genomic region of A. nidulans . The nii A and nia D genes encode nitrite and nitrate reductase respectively, and are both required for the utilization of nitrate as a nitrogen source.The nii A- nia D region was chosen after a preliminary genetic analysis of meiotic recombination, which suggested the presence of an initiation site for meiotic recombination nearby, possibly in the intergenic promoter region (chapter 2). Subsequently, we designed an assay to analyze the products of recombination in more detail. As parental strains for a series of two-point crosses, we constructed nii A and nia D mutant strains that differed by nonsense or missense mutations in the nii A and/or nia D gene and by a series of 11 silent point mutations. We analyzed recombinants selected on nitrate for the presence or absence of individual mutations to determine the position and endpoints of conversion tracts.We found evidence for an initiation site for meiotic recombination in the promoter region located between the nii A and the nia D gene. Such initiation sites have also been found in the promoter regions of several genes of S. cerevisiae. Conversion tracts with an average length of 1.5 kb extended from this site into the nii A gene, the nia D gene or both. Meiotic conversion tracts were continuous in almost all recombinants. Co-conversion of markers declined steeply with distance from the proposed initiation site. In crosses where one of the parents harbored an ectopic copy of the nii A- nia D region instead of the allelic copy, conversion tracts were similar in size and position to those observed in allelic crosses. Ectopic recombinants were detected at a significantly lower frequency. The majority of the allelic recombinants but not of the ectopic recombinants harbored a crossover within the nii A- nia D region (chapter 3).Some initial molecular genetic studies on meiotic recombination in a higher plant (maize) have recently been published. In chapter 4, we draw attention to an important similarity between meiotic recombination in fungi and maize, namely a 5' to 3' polarity of gene conversion in the loci analyzed. It is thus possible that maize, like A. nidulans and S. cerevisiae , has specific initiation sites for meiotic recombination at the 5' ends of its genes.We also examined mitotic recombination in a diploid A. nidulans strain with the same assay system (chapter 5). The observed patterns of mutations in the haploid derivatives of the mitotic recombinants suggest that the initiation site for meiotic recombination that we found in the nii A- nia D locus, does not function in mitotic recombination; furthermore, the patterns suggested that long heteroduplex tracts (> 8.4 kb) are formed during mitotic recombination, and that conversion occurs in patches within these tracts. Mitotic recombination differs in these respects from meiotic recombination in A. nidulans