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H N Arst

Publications and source records attributed to H N Arst.

At least 37 records · Page 2Linked to original sources
PubMed1997

Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA.

A change of a universally conserved leucine to valine in the DNA-binding domain of the GATA factor AreA results in inability to activate some AreA-dependent promoters, including that of the uapA gene encoding a specific urate-xanthine permease. Some other AreA-dependent promoters become able to function more efficiently than in the wild-type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild-type and mutant forms of AreA. In vitro binding studies of wild-type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild-type and mutant AreA-DNA complexes derived from the atomic coordinates of the GATA-1-AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA-binding sites, is sufficient to ensure activation of transcription of the uapA gene.

Aspergillus nidulans↗
PubMed1997

Characterization of the pH signal transduction pathway gene palA of Aspergillus nidulans and identification of possible homologs.

We have cloned the palA gene of Aspergillus nidulans, one of six genes participating in ambient pH signal transduction in a regulatory circuit mediating pH regulation of gene expression. The derived 798-residue PalA protein is 29.4% identical over its entire length to a hypothetical protein from the nematode Caenorhabditis elegans and also has possible yeast homologs.

Amino Acid Sequence↗
PubMed1996

Nitrogen metabolite signalling involves the C-terminus and the GATA domain of the Aspergillus transcription factor AREA and the 3' untranslated region of its mRNA.

AREA is a GATA transcription factor which mediates nitrogen metabolite repression in Aspergillus nidulans in response to intracellular glutamine levels. We have identified and localized three elements important to modulation of AREA function: a region of 13 residues within the DNA-binding GATA domain which forms a putative extended loop structure, the 12 C-terminal residues, and sequences within a 218 nucleotide region of the 3' UTR. The 12 C-terminal residues are also required for transcriptional activation at a subset of loci under areA control. Specific deletions within the 3' UTR and the C-terminus cause similar levels of derepression and the mutations are additive, implicating two principal signal transduction pathways. The contribution of the 3' UTR to AREA modulation is effected at the level of transcript stability such that the areA mRNA is at least five times more stable under nitrogen-derepressing conditions than it is under repressing growth conditions.

Amino Acid Sequence↗
PubMed1996

Identification, cloning and analysis of the Aspergillus niger gene pacC, a wide domain regulatory gene responsive to ambient pH.

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungus Aspergillus niger. This gene, pacC, is able to restore a pacC+ phenotype to A. nidulans pacCc11 and pacCc14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. The pacC gene of A. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream of pacC may indicate autoregulation.

Acid Phosphatase↗
PubMed1996

Translational initiation competence, 'leaky scanning' and translational reinitiation in areA mRNA of Aspergillus nidulans.

(1) AUG codons that either permit or prevent 'leaky scanning' of mRNA encoding AREA, the transcriptional activator mediating nitrogen metabolite repression in Aspergillus nidulans, have been identified. The consensus context for a strong initiation codon (i.e. one preventing 'leaky scanning') derived from this work is GXX AUG C/UCX. However, AUG codons which do not conform to this consensus are nevertheless able to initiate translation. (2) Translational reinitiation can occur within areA mRNA, although there is a limitation determined by the distance between the chain termination and reinitiation codons and/or the strength of the reinitiation codon. (3) The minimum size of the region of areA mRNA that is competent for initiation of translation has been determined to be 329 nucleotides (nt), and might be at least 975 nt if a 709 bp deletion does not alter it.

Amino Acid Sequence↗
PubMed1995

Signaling of ambient pH in Aspergillus involves a cysteine protease.

In Aspergillus nidulans, the regulation of gene expression in response to changes in ambient pH is mediated by the PacC zinc finger transcriptional regulator. At alkaline ambient pH, PacC is proteolytically processed to a functional form serving as an activator of alkaline-expressed genes and a repressor of acid-expressed genes. This activation of PacC occurs in response to a signal mediated by the products of the pal genes. Thus, the products of the palA, -B, -C, -F, -H, and -I genes constitute an alkaline ambient pH signal transduction pathway. How the pal signal transduction pathway senses ambient pH and transduces a signal to trigger PacC processing is a fascinating unresolved problem. We have cloned and sequenced the palB gene. The predicted palB gene product has similarity to the catalytic domain of the calpain family of calcium-activated cysteine proteases. We have shown, however, that the PalB protein does not catalyze the final step of proteolytic processing of PacC.

Amino Acid Sequence↗
PubMed1995

Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety.

Extremes of pH are an occupational hazard for many microorganisms. In addition to efficient pH homeostasis, survival effectively requires a regulatory system tailoring the syntheses of molecules functioning beyond the cell boundaries (permeases, secreted enzymes, and exported metabolites) to the pH of the growth environment. Our previous work established that the zinc finger PacC transcription factor mediates such pH regulation in the fungus Aspergillus nidulans in response to a signal provided by the products of the six pal genes at alkaline ambient pH. In the presence of this signal, PacC becomes functional, activating transcription of genes expressed at alkaline pH and preventing transcription of genes expressed at acidic pH. Here we detect two forms of PacC in extracts, both forming specific retardation complexes with a PacC-binding site. Under acidic growth conditions or in acidity-mimicking pal mutants (defective in ambient pH signal transduction), the full-length form of PacC predominates. Under alkaline growth conditions or in alkalinity-mimicking pacCc mutants (independent of the ambient pH signal), a proteolysed version containing the amino-terminal approximately 40% of the protein predominates. This specifically cleaved shorter version is clearly functional, both as an activator for alkaline-expressed genes and as a repressor for acid-expressed genes, but the full-length form of PacC must be inactive. Thus, PacC proteolysis is an essential and pH-sensitive step in the regulation of gene expression by ambient pH. Carboxy-terminal truncations, resulting in a gain-of-function (pacCc) phenotype, bypass the requirement for the pal signal transduction pathway for conversion of the full-length to the proteolyzed functional form.

Aspergillus nidulans↗
PubMed1995

Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.

In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources. The residual growth of the mutant strains is due to the activity of a broad specificity purine permease. We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations. Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively. The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed. uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene. The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments. The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters. Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships.

Amino Acid Sequence↗
PubMed1995

The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH.

The pH regulation of gene expression in Aspergillus nidulans is mediated by pacC, whose 678 residue-derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth at alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic glutamine repeat. Nine pacC+/- mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth and conidiation. A PacC fusion protein binds DNA with the core consensus GCCARG. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction.

Acid-Base Equilibrium↗
PubMed1995

Mutational analysis reveals dispensability of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression.

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C-terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon-encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.

Amino Acid Sequence↗
PubMed1994

Two new genes involved in signalling ambient pH in Aspergillus nidulans.

Two new genes, palH and palI, where mutations mimic the effects of acidic growth pH have been identified in Aspergillus nidulans. A palH mutation is phenotypically indistinguishable from mutations in the palA, palB, palC, and palF genes, whereas palI mutations differ only in that they allow some growth at pH 8. Mutations in palA, B, C, F, and H are epistatic to a palI mutation and the significance of this epistasis is discussed. Additionally, palE and palB mutations have been shown to be allelic. Thus, a total of six genes where mutations mimic acidic growth conditions has been identified.

Alleles↗
PubMed1994

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. alpha-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.

Arabinose↗
PubMed1994

Genetic and molecular characterisation of purine permease genes of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.

The ascomycete fungus Aspergillus nidulans can utilize purines (adenine, guanine, hypoxanthine, xanthine, and uric acid) as sole nitrogen sources [1]. The expression of most structural genes involved in the pathway of purine uptake and catabolism is subject to uric acid induction, mediated by the product of the positive regulatory gene uaY, and to nitrogen metabolite repression, mediated by the product of the general, positive-acting, GATA-like transcription factor, encoded by the areA gene [1].

Aspergillus nidulans↗
PubMed1994

Virulence studies of Aspergillus nidulans mutants requiring lysine or p-aminobenzoic acid in invasive pulmonary aspergillosis.

To identify steps in fungal intermediary metabolism required by Aspergillus spp. during invasive pulmonary aspergillosis, we have developed murine models involving Aspergillus nidulans as the inoculum. The advantages of using A. nidulans over Aspergillus fumigatus or Aspergillus flavus, which are the most common agents of clinical disease, are the well-understood genetics of A. nidulans and a large range of mutants of this species which are affected in a variety of metabolic pathways. Comparison of the virulence of A. nidulans strains carrying mutations which block the biosynthesis of lysine (lysA2) and p-aminobenzoic acid (pabaA1) shows that lysA2 strains have reduced virulence while pabaA1 strains are entirely nonpathogenic. The pathogenicity of pabaA1 strains can be restored by supplementing the drinking water of animals with p-aminobenzoic acid. The results indicate that the availability of lysine in the lung is limited, and p-aminobenzoic acid is probably not available at all. Thus, models of invasive pulmonary aspergillosis involving A. nidulans can be used to identify metabolic pathways that may be essential for the pathogenicity of A. fumigatus, the predominant pathogenic species, suggesting potential new targets for antifungal therapy.

4-Aminobenzoic Acid↗
PubMed1993

Characterization of an Aspergillus nidulans genomic DNA fragment conferring phosphate-non-repressible acid-phosphatase activity.

A clone from an Aspergillus nidulans library was identified by its ability to confer enhanced staining for acid phosphatase (APase) activity upon phosphatase-deficient A. nidulans mutants. This APase activity is not repressed by high phosphate concentrations in the medium. The 2.9-kb nucleotide sequence in the region of the clone responsible for the effect reveals two potential protein-coding genes with a common N terminus. One corresponds to an open reading frame (ORF) with no introns, encoding 330 amino acids (aa). The other, shorter gene encoding 113 or 117 aa has the first 65 or 69 codons in common with the long ORF; then, after a single 165-nt intron with a fungal consensus lariat sequence and splice junctions, there are a further 48 codons in a different reading frame. Both correspond in sense direction, and the shorter gene in length, with the only detectable transcript in this region, but both differ from all known APase sequences. The possible identity of these ORFs with the pacG gene is discussed.

Acid Phosphatase↗