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.     

Aspergillus nidulans polarity mutant swoA is complemented by protein O-mannosyltransferase pmtA

Previously swoA was identified in Aspergillus nidulans as a single locus, temperature-sensitive (ts) mutant aberrant in polarity maintenance. swoA was complemented by transformation with a plasmid genomic library. The sequence of the complementing gene was identical to a previously submitted GenBank sequence for pmtA, a protein O-mannosyltransferase. The pmtA/swoA-2 gene hybridized to three cosmids that are located on chromosome V and the swoA mutation was mitotically mapped to chromosome V. PMTs are endoplasmic reticulum-resident proteins responsible for the first step of O-glycosylation. Structural predictions suggest that PmtA contains seven membrane spans similar to PMTs from Saccharomyces cerevisiae and other organisms. Phylogenetic analysis indicates that PmtA is most closely related to the S. cerevisiae sub-family of PMTs containing Pmt2, Pmt3 and Pmt6. The mutant pmtA/swoA-2 locus contained a lesion that changed Y662 to a stop codon, eliminating the final membrane spanning domain and interrupting a domain essential for function in other PMTs.     

An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity

Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life-threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase-chain-reaction-based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS-polyacrylamide gel electrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild-type cells, the mutant had undetectable activity on azocollagen and elastin-Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.     

A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation

Upon germination Aspergillus nidulans swoM1 exhibits abnormal development by extending a primary germ tube that quickly reverts to isotropic growth and results in an enlarged, swollen apex with pronounced wall thickenings. Apical lysis occurs in 38% of the germlings. A point mutation in the AN6037.3 gene encoding the only phosphoglucose isomerase in A. nidulans is responsible for the defect. Loss of polarity is bypassed when glucose is replaced with alternate carbon sources but in all cases the mutant is unable to conidiate due to a block in conidiophore development at vesicle formation. In conidiophores SwoM::GFP localizes to multiple punctate, foci within each actively growing cell type, and to multiple foci in mature dormant conidia. In hyphae SwoM::GFP localized to two rings spanning the center of mature septa. In hyphae localization is concentrated at actively growing hyphal tips.     

Asexual recombination in a uvsH mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.     

Characterization of an Aspergillus nidulans L-arabitol dehydrogenase mutant

Abstract The degradation pathway for L-arabinose, which consists of a sequence of alternating reduction and oxidation reactions prior to ultimate phosphorylation, was studied in Aspergillus nidulans wild-type as well as in an L-arabinose non-utilizing mutant. The inability of the mutant to use L-arabinose was caused by the absence of L-arabitol dehydrogenase activity. The effect of the mutation on polyol accumulation patterns was studied upon growth on various carbon sources. The presence of L-arabinose resulted in intracellular accumulation of arabitol in this mutant. Moreover, the mutant secreted arabitol under these conditions and, in contrast to the wild-type, featured enhanced expression of enzymes involved in L-arabinose catabolism as well as of extracellular glycosyl hydrolases involved in degradation of the plant cell wall polysaccharide L-arabinan.     

UV-induced DNA degradation in Aspergillus nidulans cells]

UV-induced DNA degradation was studied in mycellial cells of Aspergillus nidulans wild type and several uvs mutants. It was shown to be an enzymatic specific process which possibly reflects the excision of pyrimidine dimers from UV-damaged DNA. Inhibition of DNA degradation by caffeine and 2,4-dinitrophenol shows the connection between degradation and repair of DNA. Two ways of DNA degradation were found in A. nidulans cells, one of them being glucose dependent and the other--glucose independent. The dependence of DNA degradation on protein synthesis before and after UV-irradiation was demonstrated. The scheme of ways of DNA degradation and its genetic control were suggested on the basis of uvs mutations effect on UV-induced DNA degradation.     

The uvsC and uvsE genes of Aspergillus nidulans are not required for the mutagenic repair of UV damage

The uvsC gene of Aspergillus nidulans is a homolog of the RAD51 gene of Saccharomyces cerevisiae. However, with respect to its effects on UV mutagenesis, it differs from the yeast gene, since it seems to be required for UV mutagenesis; however, this conclusion is based only on data from resting conidia. To further clarify the functional role of the uvsC gene, we tested the UV mutability of strains bearing a uvsC mutation in resting as well as in germinating conidia, by the p-fluoro-phenyl-alanine resistance test. We also evaluated the mutability of the uvsE mutant which belongs to the same epistatic group. Our results show that the uvsC and uvsE genes do not have a significant role in the mutagenic UV-repair pathway.     

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.     

A mycobacterial smc null mutant is proficient in DNA repair and long-term survival

SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for smc null mutants in other species.     

A mutant of Aspergillus nidulans defective in NAD-linked glutamate dehydrogenase.

A mutation leading to partial loss of NAD-linked ("catabolic') glutamate dehydrogenase does not affect the regulation of ammonium-repressible activities in Aspergillus nidulans. This mutation has been used to show that NAD-linked glutamate dehydrogenase does not normally participate in ammonium assimilation. A mutation leading to loss of NADP-linked ("anabolic') glutamate dehydrogenase has been used to show that NADP-linked glutamate dehydrogenase is not normally involved in glutamate catabolism. Strains defective in either enzyme are useful for determining which amino acids are metabolised via transamination to yield glutamate rather than via deamination to yield ammonium.