The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

Dynamic association of topoisomerase II to the mitotic chromosomes in live cells of Aspergillus nidulans

DNA topoisomerase II (Topo II) is an essential enzyme that catalyzes topological changes of DNA and consists of a major member of mitotic chromosomes. To investigate the dynamic localization of Topo II in nuclei, we engineered the strain of Aspergillus nidulans expressing Topo II fused with green fluorescent protein (GFP). Time-lapse microscopy revealed that the distribution of Topo II-GFP in nuclei varied depending on the cell cycle. In interphase, Topo II-GFP distributed evenly in the nucleoplasm and at the onset of G2 phase became concentrated into nucleolus. During mitosis, Topo II-GFP accumulated on chromosomes, when the chromosomes condensed. In the early mitosis, the Topo II also showed a single or two brighter spots among the fluorescence of clumped chromosomes. The spots once divided into several spots and then concentrated again into a spot per nucleus in the dividing nuclei of anaphase. Along with the subsequent decondensation of chromosomes, Topo II diffused back into nucleoplasm.     

Evidence for carbon source regulated protein kinase A and protein kinase C signaling in the duplication cycle,polarization and septum formation in Aspergillus nidulans

The effects of glucose and of a pectic substrate in the duplication cycle, spore polarization and septation of Aspergillus nidulans were tested in poor and rich media. Growth on poor conditions and on sodium polypectate slowed nuclear duplication and reduced the coupling of polarization to mitosis. Coupling of septation to the third mitosis was also reduced by changing growth conditions. When protein kinase A (PKA) and protein kinase C (PKC) activators were added to the media the results suggested a role for PKA in slowing the duplication cycle, while allowing polarization. Addition of a PKC activator to poor media uncoupled the first septum formation from the third mitosis in a carbon source-regulated manner, suggesting a role for PKC in coordinating cell cycle signals, growth and cytokinesis.     

Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.     

Genetical and biochemical aspects of quinate breakdown in the filamentous fungus Aspergillus nidulans

In the ascomycetous fungus Aspergillus nidulans, the expression of two inducible, contiguous or closely linked genes (qutB and qutC) which encode enzymes for quinate breakdown to protocatechuate, appears to be controlled by the product of a tightly linked third gene (qutA). The qut gene cluster locates on chromosome VIII. The catalytic steps required for this conversion are dehydrogenase, dehydroquinase, and dehydratase, and these activities are induced by the presence of quinate in a similar manner. The dehydroquinase enzyme has been purified and shown to be multimeric, consisting of 20–22 identical subunits of approximately 10,000 MW. The enzyme has a pI value of 5.84, a K _m of 5×10^–4 m, and an amino acid composition that lacks tryptophan and cysteine. The enzyme also cross-reacts with rabbit antibodies raised against Neurospora crassa catabolic dehydroquinase.This work was supported in part by European Molecular Biology Organisation grants to J.R.K. and A.R.H. and by National Institutes of Health Grant GM23051 to N.H.G.     

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular l-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. α-l-Arabinofuranosidase B, endoarabinase, l-arabinose reductase, l-arabitol dehydrogenase, xylitol dehydrogenase, and l-xylulose reductase were all inducible to varying degrees by l-arabinose and l-arabitol and subject to carbon catabolite repression by d-glucose. With the exception of l-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on d-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of “self” catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.     

Proteome analysis of the farnesol-induced stress response in Aspergillus nidulans--The role of a putative dehydrin

The isoprenoid alcohol farnesol represents a quorum-sensing molecule in pathogenic yeasts, but was also shown to inhibit the growth of many filamentous fungi. In order to gain a deeper insight into the antifungal activity of farnesol, we performed 2D-differential gel electrophoretic analysis (2D-DIGE) of Aspergillus nidulans exposed to farnesol. We observed an increased abundance of antioxidative enzymes and proteins involved in protein folding and the ubiquitin-mediated protein degradation. A striking finding was the strong up-regulation of a dehydrin-like protein (DlpA). Expression analyses suggested the involvement of DlpA in the cellular response to oxidative, osmotic and cold stress. In line with these data, we demonstrated that dlpA expression was regulated by the MAP kinase SakA/HogA. The generation of both a dlpA Tet(on) antisense RNA-producing A. nidulans strain (dlpA-inv) and a ΔdlpA deletion mutant indicated a role of DlpA in conidiation and stress resistance of dormant conidia against heat and ROS. Furthermore, the production of the secondary metabolite sterigmatocystin was absent in both strains dlpA-inv and ΔdlpA. Our results demonstrate the complexity of the farnesol-mediated stress response in A. nidulans and describe a farnesol-inducible dehydrin-like protein that contributes to the high tolerance of resting conidia against oxidative and heat stress.     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans

Summary We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.Abbreviations FPA DL-p-fluorophenylalanine - phenA auxotroph of phenylalanine - Phe-tRNA synthetase phenylalanyl-transfer ribonucleic acid synthetase Current address: Department of Biological Sciences (M/C 066) The University of Illinois at Chicago, Box 4348, Chicago, IL 60680, USA     

Expression of a synthetic Artemesia annua amorphadiene synthase in Aspergillus nidulans yields altered product distribution

A gene encoding a plant terpene cyclase, Artemisia annua amorpha-4,11-diene synthase (ADS), was expressed in Aspergillus nidulans under control of a strong constitutive promoter, (p)gpdA. The transformants produced only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in planta. In contrast, expression of ADS in Escherichia coli produced almost exclusively amorpha-4,11-diene. These results indicate that the host environment can greatly impact the terpenes produced from terpene synthases.     

Regulation of gene expression by pH of the growth medium in Aspergillus nidulans

Summary In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for -amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pH_in) which is more alkaline than that of the wild type whereas pacC mutations result in a pH_in more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.These genes are, to our knowledge, the first examples of genes involved in the regulation of extracellular enzyme and permease synthesis by the pH of the growth medium to be described in any organism.     

Sequence, organization and expression of the core histone genes of Aspergillus nidulans

Summary The core histone gene family ofAspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in theA. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 by and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established forA. nidulans. This karyotype was used to assign the H3–H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A–H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.     

csmA,a gene encoding a class V chitin synthase with a myosin motor-like domain of Aspergillus nidulans,is translated as a single polypeptide and regulated in response to osmotic conditions

The csmA gene of Aspergillus nidulans encodes a polypeptide that consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain. csmA null mutants showed marked abnormalities in polarized growth, hyphal wall integrity, and conidiophore development. Furthermore, the growth of the csmA null mutants was sensitive to low osmotic conditions. In an effort to investigate the intracellular behavior of the csmA product (CsmA) and the regulation of its production, we constructed strains that produced CsmA tagged with nine repeats of the hemagglutinin A (HA) epitope at its COOH terminus (CsmA-HA) instead of CsmA. Western blot analysis with anti-HA antibody showed that the entire coding region of csmA was translated as a single polypeptide with an approximate molecular mass of 210kDa. CsmA-HA was produced during vegetative growth; however, its yield was significantly reduced under high osmotic conditions, suggesting that the role of CsmA in growth and morphogenesis is particularly important under low osmotic conditions.