Genetic analysis in the asexual fungus Aspergillus niger.

The genetics of A. niger has been developed since 1980. An overview is presented of the advances in developing methods and collecting data. Important tools have been a) the application of essentially different methods to isolate mutants, b) the adaptation to A. niger ofA. nidulans methodology for analysis of the parasexual cycle, c) the choice of marker genes, and in some cases the artificial introduction of such genes, to select homozygous segregants arising from mitotic recombination. With the use of parasexual recombination, a genetic linkage map of A. niger has been established. In total, 110 nuclear and 1 cytoplasmic (mitochondrial) markers are available. The application of A. niger genetics in applied research is illustrated by examples.

Genetic analysis of resistance to fenpropimorph in Aspergillus niger.

Resistance to the morpholine-fungicide fenpropimorph was studied in Aspergillus niger and A. nidulans. Mass selection of conidia of A. nidulans on agar amended with the fungicide at different concentrations did not yield of resistant mutants, even after UV-treatment of the conidia. In contrast, similar experiments with A. niger generated many fenpropimorph-resistant mutants. The mutants displayed cross-resistance to fenpropidin and generally showed wild-type sensitivity to the unrelated toxicants fenarimol and cycloheximide. Genetic analysis of fenpropimorph resistance in A. niger was carried out by means of the parasexual cycle. In the mutants tested, two genes located on linkage group II were involved in fenpropimorph resistance. Dominance tests showed that resistance to fenpropimorph in A. niger is recessive.

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

An electrophoretic karyotype of Aspergillus niger.

An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosome-sized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage group-specific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5-38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping.

The Aspergillus nidulans gene coding for acetamidase (amdS) was introduced into A. niger by transformation. Twelve Amd+ transformants were analysed genetically. The amdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed) A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for the amdS insert have the Amd+ phenotype. We exploited the opportunity for two-way selection in A. niger: transformants can be isolated based on the Amd+ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologous amdS gene in A. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI between pabA1 and cnxA1, providing evidence for a single transformational insert. The results also show that the amdS transformants of A. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use of amdS transformants for mitotic gene mapping in other fungi.