Microheterogeneity in Aspergillus nidulans 5S rRNA genes

Summary We have determined the sequence of 4 Aspergillus nidulans 5S rRNA genes and compared it with 4 previously established sequences. No extensive homologies are found in 5 flanking sequences, but in the 3 flanks of two genes and two pseudogenes similar sequences are observed. In the coding sequences differences occur in 7 positions. Two 5S rRNA genes which are found in one plasmid 1.1 kb apart are located in opposite orientations.     

Suppressor specificity in Aspergillus nidulans

Summary Seven allele specific gene unspecific suppressors mapping at four loci have been described previously (Roberts et al. 1979). Three new suppressors mapping in suaA are characterised, and the spectrum of suppression of all the suppressors with respect to seventeen suppressible mutations in eight different genes is described. Two distinct classes of suppressor are defined. The diversity of suppression of five suaA alleles, and the temperature sensitivity of some suaA suppressor mutant combinations but not others, suggests that suppressors at this locus are acting via ribosomal protein alteration. suaC109, a mutation that results in cold-sensitivity for growth shows a similar broad spectrum of suppression. Suppressors at the suaA and suaC loci suppress mutations that have the properties of chain termination mutations as well as missense mutations. suaB111, and suaD103 and suaD108 have a very restricted range of suppression. These suppressors may be mutations in tRNA genes.     

Heat shock phenomena in Aspergillus nidulans

Summary Heat shock was found to induce characteristic changes in the pattern of protein synthesis in Aspergillus nidulans as analysed by SDS-polyacrylamide gel electrophoresis. Six to seven new bands were found to show increased incorporation to ³⁵S-methionine at 43 °C compared to 37 °C, the standard temperature for this organism. The heat shock response of five different strains of A. nidulans was examined. This comparative study showed that these strains (haploids and diploids) show exactly the same set of heat shock proteins.     

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans

Summary Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.Deceased April 30, 1988     

The identification of mutations in Aspergillus nidulans that lead to increased levels of ADHII

Summary There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHII has been observed in polyacrylamide gels stained for ADH activity but, unlike ADHI and ADHIII, no physiological function has been attributed to it. This paper describes mutations that have been isolated from strains carrying a deletion in the structural gene for ADHI (alcA) and its adjacent positively-acting regulatory gene (alcR) that restore some ability to utilise ethanol as a carbon source. The mutations map at three loci, and all show elevated levels of the ADHII staining band. An assay for ADHII has been developed. The growth on ethanol has been shown to be dependent on the previously identified aldehyde dehydrogenase (structural gene, aldA). Two of the mutations, alcD and alcE, represent newly discovered mutations affecting ethanol utilisation while the third mutation is in amdA, a previously described trans-acting regulatory protein.     

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Antisuppressor mutations reduce misreading of the genetic code in Aspergillus nidulans

Summary Antisuppressor mutations were isolated in a strain containing the omnipotent suppressor suaC109. The antisuppressors reduce the activity of translational suppressors in vivo and counteract most aspects of the pleiotropic phenotype associated with the suaC and the suaA suppressor mutations. Using an homologous system for cell-free translation, we have measured translational accuracy in two antisuppressor strains with the genotype suaC109 and either the asuB11 or the asuD14 antisuppressor mutation. Ribosomes from antisuppressor mutants have higher levels of translational accuracy than those from the suppressor strain (suaC109, asu ⁺). Mistranslation levels depended solely on the source of the sucrose-cleaned ribosomes. However, the increased accuracy associated with sucrose-cleaned ribosomes from antisuppressor strains can be nullified by salt-washing, suggesting that the component responsible can be washed off.     

Every ribosomal suppressor mutation in Aspergillus nidulans has a unique and highly pleiotropic phenotype

Summary 18 suppressors of alcR125 have been selected in Aspergillus nidulans. They have been located in genes as follows: 12 in suaA, 1 in suaB and 5 in suaC. Suppressors have been examined to see whether their phenotype is diagnostic for their genotype. Several new traits are described: conidial viability, cycloheximide resistance, fertility, suppression of niaD500, naaD501 and fWA1. These tests, added to those already in use, provide a battery of tests suitable for assigning suppressor mutations to physiological type (tRNA or ribosomal), and in one case to a specific gene since only suaA mutations suppressed fWA1. A very broad range of phenotypes was associated with suppressors such that every mutation had a unique phenotype. This indicates that the ribosomal suppressor mutations are in genes which code directly for ribosomal proteins, rather than genes which code for modifying enzymes.     

Hygromcyin- and paromonycin-resistant mutants of Aspergillus nidulans alter translational fidelity

Summary Mutants of Aspergillus nidulans resistant to the aminoglycoside antibiotics paromycin and hygromycin B have been isolated and their growth characteristics are described here. Most paromomycin mutants were crossresistant to hygromycin and geneticin. All the hygromycin-resistant mutants were slightly cross-resistant to geneticin. Out of the 15 mutants tested 14 had drug-resistant ribosomes in vitro and all 12 of those investigated further had reduced levels of translational misreading. Five new loci have been found-parA on linkage group I, hygA on III, hygB on IV, hygC on V, hygD on VI and parB on VIII. This increases, to at least 12, the number of translational fidelity loci in A. nidulans.Amina Sheikh is the nee Zamir     

Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans

Summary Thirty-three argB^– to argB⁺ transformants of Aspergillus nidulans have been subjected to genetic and molecular analysis. Two showed high levels of mitotic instability although it is suggested that this is a consequence of heterokaryosis rather than instability of the transformation event. Most transformants resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus. The maximum number of inserted sequences was two, to generate three copies of the argB locus. The other main transformant type showed replacement of the argB^– mutation by the wild-type allele present on the transforming plasmid. Transformants were also recovered in which the transforming DNA had integrated into non-homologous chromosomal regions. Selfed or hybrid cleistothetica from all transformants, except the gene replacement types gave arginine requiring recombinants. Most transformants showed low levels of meiotic instability. Others displayed varying levels which in some cases differed between selfed and hybrid cleistotheticia. There was some correlation between meiotic instability and the nature of the transformation event. Diploid parasexual and aneuploid analysis located the integrated DNA in each transformant to chromosome III. Two transformants were isolated as heterozygous diploids. A third diploid was isolated as a stable mitotic segregant from one of the mitotically unstable transformants.     

Heat shock phenomena in Aspergillus nidulans

Summary The combined action of hyperthermia and Bleomycin on Aspergillus nidulans was studied at three different levels: mycelial protein synthesis, spore viability and induction of mutations. It was found that Bleomycin treatment of preincubated mycelia during the heat shock enhances the incorporation of ³⁵S-methionine into heat shock bands. Furthermore, simultaneous treatment with hyperthermia (43°C) and Bleomycin results in greater cytotoxic activity in spores and in a higher induction rate of point mutations.     

Two family G xylanase genes from Chaetomium gracile and their expression in Aspergillus nidulans

With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.     

Domain-wide,locus-specific suppression of nitrogen metabolite repressed mutations in Aspergillus nidulans

Summary Previous work has shown that loss of function mutations (designated are A^r) in areA, a positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans, lead to inability to utilise nitrogen sources other than ammonium. This work establishes the existence of a gene designated areB where mutations can suppress areA^r mutations in a locus-specific manner for expression of apparently all of the genes under areA control. areB mutations are partially dominant in diploids, extremely rare and, to varying degrees, deleterious. In agreement with their ability to suppress areA^r mutations, areB mutations lead to nitrogen metabolite derepressed enzyme levels. areB is located about 5 cM from creA on the left arm of linkage group I. Four of the five areB mutations selected are accompanied by translocations. In at least two of these cases the areB mutations are extremely tightly linked to and probably identical with the translocation breakpoints. These two translocations, although induced in different strains in separate experiments, apparently have almost identical breakpoints. This suggests that chromosomal rearrangements of a highly specific nature can give rise to an areB mutant phenotype.     

Molecular karyotype alterations induced by transformation in Aspergillus nidulans are mitotically stable

Clamped homogeneous electric field (CHEF)-gel electrophoresis was used to define the electrophoretic molecular karyotype of Aspergillus nidulans strain OC-1 before and after protoplast-based genetic transformation. The transforming DNA caused alterations in the molecular karyotypes in all transformants examined. Rather dramatic changes were observed in karyotypes, including apparent chromosome loss, massive size alterations, and the appearance of large chromosomes. Changes in molecular karyotype were mitotically stable.     

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins

Summary It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS⁺ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS⁺ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS⁺ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.     

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants

Summary The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.     

p-fluoro-phenylalanine resistance in Aspergillus nidulans diploid cells: evidence that dominant,lethal mutations are involved

Summary An unexpectedly large number of p-fluorophenylalanine (FPA)-resistant mutants have been recovered after UV-irradiation of wild type diploid conidia of Aspergillus nidulans. At least five different classes of mutants, possibly corresponding to five different loci, have been identified. Two of them may be the dominant loci which have already been described but the others (a minimum of three loci) are completely different. Mutations in these loci confer high level FPA resistance in the heterozygous diploids, being lethal in the haploids; one mutation has been preliminarily mapped to chromosome I and another to chromosome III.     

Ammonium ion sensitivity is a ribosomal phenotype associated with suppressor mutations in the suaC gene of Aspergillus nidulans

Summary Ammonium ions are selectively toxic to strains containing mutations in the suaC gene which can mutate to a suppressor phenotype. This phenotype is associated with increased ribosomal misreading in vitro (Zamir and Martinelli 1987) and altered ribosomal proteins (Harvey and Martinelli 1983). Such ammonium-sensitivity is a feature of both strong and weak suppressor alleles, and segregates with suppressor ability in crosses. Suppressor mutations in the suaB and suaD genes are not affected, nor are those in suaA, another ribosomal suppressor gene. Thus, the ammonium-effect is locus specific. Mutations which act as antisuppressors (asu ^–) of suppressor suaC109 also partially reverse the ammonium ion sensitivity associated with this mutation. This effect is in line with their restoration of other aspects of the pleiotropic phenotype to normal. The cations, lithium and rubidium, mimick the effects of ammonium ions. Only ribosomes from suaC strains are sensitive to the presence of NH ₄ ⁺ ions in vitro.     

The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity

The protein kinase C (PKC) family participates in maintaining integrity and growth of fungal cell walls. However, the precise molecular role of these proteins in the filamentous fungi remains unknown. In this work, pkcA, the gene encoding the PKC homolog in the filamentous fungus Aspergillus nidulans, was cloned and its function analyzed using a conditional alcA-PKC mutant strain. Repression of pkcA expression resulted in increased conidial swelling, decreased rates of hyphal growth, changes in the ultrastructure of the cell wall and increased sensitivity to antifungal agents. These results suggest that the protein encoded by pkcA is involved in key aspects of cell morphogenesis and cell wall integrity.