Chromosomal mapping of an alcC disruption with respect to amdA in Aspergillus nidulans

Summary We report the use of the riboB gene for a gene replacement in the alcC gene of Aspergillus nidulans, and show by reverse genetics that the alcC gene is very closely linked to the amdA gene.     

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     

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

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.     

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     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

An adaptive response to alkylating agents in Aspergillus nidulans

Summary The analysis of mitochondria' DNA (mtDNA) from several strains of Candida parapsilosis and Candida rhagii by restriction endonucleases enabled us to discriminate between several groups within the C. parapsilosis species and to allocate laboratory strains to one of these. The mtDNAs isolated ranged in size from 20 to 31 kb. The mtDNA isolated from group 1 C. parapsilosis hybridises with both ATPase subunit 6 and 8 gene probes, the same restriction fragment hybridising with both probes.     

Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption

Summary A transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ^- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.     

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.     

Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans

Summary Penicillium nalgiovense was transformed with the amdS gene from Aspergillus nidulans as a selectable marker. The vector apparently integrated at multiple sites into the chromosomes of the transformants, which were mitotically stable. A transformation efficiency of 12 transformants/g vector DNA was achieved when the expression phase was prolonged to 8 h.