Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique

In the heterokaryon rescue technique, gene deletions are carried out using the pyrG nutritional marker to replace the coding region of target genes via homologous recombination in Aspergillus nidulans. If an essential gene is deleted, the null allele is maintained in spontaneously generated heterokaryons that consist of two genetically distinct types of nuclei. One nuclear type has the essential gene deleted but has a functional pyrG allele (pyrG+). The other has the wild-type allele of the essential gene but lacks a functional pyrG allele (pyrG-). Thus, a simple growth test applied to the uninucleate asexual spores formed from primary transformants can identify deletions of genes that are non-essential from those that are essential and can only be propagated by heterokaryon rescue. The growth tests also enable the phenotype of the null allele to be defined. Diagnostic PCR can be used to confirm deletions at the molecular level. This technique is suitable for large-scale gene-deletion programs and can be completed within 3 weeks.     

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.     

Isolation of nuclei from Aspergillus nidulans protoplasts

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological state, as demonstrated by fluorescence microscopy and electronmicroscopy. The weight ratio of DNA:RNA:protein was 1:3.0:10.8 in the nuclear fraction.     

Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

Isolation and Characterization of Sexual Spore Pigments from Aspergillus nidulans

The homothallic ascomycete Aspergillus nidulans produces two types of pigmented spores: conidia and ascospores. The synthesis and localization of the spore pigments is developmentally regulated and occurs in specialized cell types. On the basis of spectroscopic evidence, we propose that the major ascospore pigment of A. nidulans (ascoquinone A) is a novel dimeric hydroxylated anthraquinone. The structure of ascoquinone A, as well as a comparison to model compounds, suggests that it is the product of a polyketide synthase. Previous studies have revealed that the conidial pigments from A. nidulans and a related Aspergillus species (A. parasiticus) also appear to be produced via polymerization of polyketide precursors (D. W. Brown, F. M. Hauser, R. Tommasi, S. Corlett, and J. J. Salvo, Tetrahedron Lett. 34:419-422, 1993; M. E. Mayorga and W. E. Timberlake, Mol. Gen. Genet. 235:205-212, 1992). The structural similarity between the ascospore pigment and the toxic anthraquinone norsolorinic acid, the first stable intermediate in the aflatoxin pathway, suggests an evolutionary relationship between the respective polyketide synthase systems.     

A chromosome assay method for the detection of heterokaryon incompatibility (het) genes operating between members of different heterokaryon compatibility (h-c) groups in Aspergillus nidulans

Protoplast fusion has made possible the isolation of a diploid strain from haploid parents belonging to heterokaryon compatibility (h-c) groups Q and Gl of Aspergillus nidulans. This diploid was not fully heterokaryon compatibility tests conducted between selected pairs of parasexually derived progeny strains facilitated a chromosome assay method for the detection of heterokaryon incompatibility (het) genes. Despite the lack of segregation for the linkage group VI marker, it proved possible to locate het genes on linkage groups III, V, VI and VII. Backcross data detected five het gene differences operating between the h-cQ and h-cGl parental strains. Two het loci were located on linkage group III.     

Transformation by integration in Aspergillus nidulans

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.     

Genetic manipulation of Aspergillus nidulans: heterokaryons and diploids for dominance, complementation and haploidization analyses

The haploid microbial eukaryote Aspergillus nidulans is a powerful genetic system, which allows analysis of a broad range of biological phenomena. In addition to conventional analysis of meiotic progeny in a single generation, parasexual analysis affords a rapid and convenient method for genetic analysis. We describe the construction of A. nidulans heterokaryons and diploids for use in genetic analysis to determine dominance and conduct complementation tests. We also describe the rapid mapping of mutations to chromosomes by haploidization of diploids carrying marked chromosomes. Balanced heterokaryons may be established within 10 days and diploids may be constructed in 2-3 weeks. Dominance tests and complementation tests using balanced heterokaryons or diploids may be completed in 2-3 days. Haploidization analysis of heterozygous diploids can be achieved within 10 days. These protocols should be adaptable for use in related Aspergilli and Penicillia, which lack a known meiotic cycle.     

Transformation of Aspergillus nidulans by the argB gene

Aspergillus nidulans argB mutant was transformed with the plasmid DNA containing the argB gene. Analysis of transformants revealed that transformation was due to integration of either argB gene alone or the whole plasmid DNA into the A. nidulans genome. In 5 out of 23 transformants studied, integration took place in the locus different than the original argB locus. The amplification of integrated sequences was often observed. Integrated DNA was found to be mitotically stable, while the meiotic stability depends on the mode of integration. The activity of the ornithine carbamoyltransferase (the argB gene product) was measured and in some transformants bearing the amplified argB sequence was found to be strongly elevated.