Mutations to constitutivity and derepression are separate and separable in a regulatory gene of Aspergillus nidulans

Summary Previous work has shown that expression of the structural genes for the enzymes of nitrate and nitrite assimilation in Aspergillus nidulans requires the products of two positively acting regulatory genes — nirA, mediating induction, and areA, mediating nitrogen metabolite repression. Here we show that, in addition to previously described mutations in nirA leading to constitutivity, other mutations can be selected in nirA leading to nitrogen metabolite derepression. These constitutivity and depression mutations in nirA are additive and separable by intragenic recombination. This suggests that the nirA gene product contains two separate domains, a co-inducer binding region, defined by constitutivity mutations, and a region interacting with the areA gene product or with initiator sites adjacent to structural genes under areA and nirA control, defined by derepression mutations. These findings might indicate a striking similarity of action between the eukaryotic regulatory gene nirA and a comparable prokaryotic regulatory gene.     

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.     

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.     

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     

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.     

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.     

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.     

Nitrogen metabolite repression in Aspergillus nidulans: A farewell to tamA?

Summary Previous work has established that nitrogen metabolite repression in Aspergillus nidulans is mediated by the positive acting regulatory gene areA. Pateman and Kinghorn (1977) proposed that the gene tamA plays an equally important regulatory role in nitrogen metabolite repression as the result of work with tamA^r-50, an allele leading to inability to utilise nitrogen sources other than ammonium, and tamA^d-1, an allele leading to nitrogen metabolite derepression. Both tamA^r-50 and tamA^d-1 were subsequently lost. We have therefore attempted to reconstruct Pateman and Kinghorn's work with tamA. We propose that tamA^r-50 was in fact a pyroB^– tamA^– double mutation. pyroB^– mutations lead to a block in vitamin B₆ biosynthesis which can be supplemented by extremely high concentrations of ammonium. tamA^– mutations, possibly as the result of a membrane alteration, reduce the concentration of ammonium required to supplement the pyroB^– auxotrophy. There is, however, no evidence that pyroB^– or tamA- mutations, alone or in combination, affect the regulation of the levels of a number of enzymes subject to nitrogen metabolite repression. Reversion of pyroB^– strains constitutes a powerful positive selection technique for obtaining a wide variety of mutations in glnA, the probable structural gene for glutamine synthetase. We suggest that the nitrogen metabolite derepressed phenotype attributed to tamA^d-1 might have resulted from an extremely leaky glnA^– mutation.     

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.     

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library

Autonomously-replicating, marker-less helper plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.     

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.     

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.     

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.     

Additive action of partial heterokaryon incompatibility (partial-het) genes in Aspergillus nidulans

We have observed partial heterokaryon-incompatibility reactions in combinations of field isolates of A. nidulans. We have demonstrated that partial heterokaryon incompatibility is genetically controlled by genes (partial-het genes) operating in the same manner as the previously-described het genes. Our results also reveal that partial-het genes can act additively in causing heterokaryon incompatibility and that partial heterokaryon incompatibility is not a barrier to the horizontal transfer of a mitochondrial marker. These results add to the growing body of evidence that vegetative-incompatibility reactions are not an absolute barrier to horizontal gene flow.     

A mitochondrial reading frame which may code for a second form of ATPase subunit 9 in Aspergillus nidulans

Summary The nucleotide sequence of a 74 codon reading frame from the Aspergillus nidulans mitochondrial genome is presented. The derived amino acid sequence displays typical features of dicyclohexylcarbodiimide (DCCD) binding proteins and is 84% homologous with a mitochondrial reading frame that potentially encodes an ATPase subunit 9 polypeptide in Neurospora crassa. However, in A. nidulans, as in N. crassa, there is strong biochemical and genetic evidence that this subunit is in fact nuclearly-encoded. In both organisms the DCCD-binding protein found in the F₀ complexes of mitochondria from actively-growing cultures is almost certainly the product of this nuclear gene, and definitely not that of the mitochondrial reading frame. The discovery of an intact open reading frame than can code for a DCCD-binding protein in the mitochondrial genome of a second species of filamentous fungus strenghthens the possibility that the presence of a mitochondrial version of this gene has some biological significance.     

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.     

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     

The cloning and sequencing of thealcB gene,coding for alcohol dehydrogenase II,inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in gt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5 region of the gene is discussed in relation to the regulation of the gene.     

Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system

The ability to perform multiple gene deletions is an important tool for conducting functional genomics. We report the development of a sequential gene deletion protocol for the filamentous fungus Aspergillus nidulans using the Cre/loxP recombinase system of bacteriophage P1. A recyclable genetic marker has been constructed by incorporating loxP direct repeats either side of the Neurospora crassa pyr-4 gene (encodes orotidine 5′-monophosphate decarboxylase) which is able to complement the A. nidulans pyrG89 mutation. This construct can be directed to delete specific genomic regions by attaching flanking sequences corresponding to the desired target. The pyr-4 marker can subsequently be eliminated by Cre-catalysed recombination between the loxP sites. The recombinase gene (cre), which has been placed under the control of the A. nidulans xlnA (xylanase A) gene promoter thus providing a means to switch on (xylose induction) or off (glucose repression) recombinase expression, has been integrated into the genome of an A. nidulans mutant strain defective in orotidine 5′-monophosphate decarboxylase activity (pyrG89). We demonstrate the effectiveness of our deletion system by sequentially deleting two genes, yellow (yA) and white (wA), involved in the synthesis of conidial pigment.