A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus.

Ochratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1.4 kb clone shared 28-35 % identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.

Differential expression of cold- and diet-specific genes encoding two carp liver delta 9-acyl-CoA desaturase isoforms.

Carp respond to cold by the upregulated expression of Delta9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second Delta9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of delta9-monounsaturates, confirming their identity as delta9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30 degrees C to 10 degrees C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15 degrees C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of delta9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment.

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA.

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3'-UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP-linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation.

In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA-binding proteins. In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss-of-function mutations. Based on our analysis, areB is predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non-AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4). We have characterized three classes of mutation in areB; the first are loss-of-function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative-acting transcription factor into an activator that can partially replace areA.

Evaluating low level sequence identities. Are Aspergillus QUTA and AROM homologous?

A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7-18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene 136, 49-54]. We tested this hypothesis by a sensitive combination of position-specific log-odds scoring matrix methods. The position-specific log-odds scoring matrices were derived from a large number of 3-dehydroquinate synthase and 5-enolpyruvylshikimate-3-phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position-specific log-odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA-AROM homology even more problematic.

A defined sequence within the 3' UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation.

Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3' untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

Thermal thresholds of lipid restructuring and delta(9)-desaturase expression in the liver of carp (Cyprinus carpio L.).

Cold acclimation induces a transient enzymatic activation of the acyl CoA-(&Dgr;)(9)-desaturase in carp liver. We have determined thresholds for two underlying mechanisms; namely, the activation of latent enzyme and the induced synthesis of new desaturase. Carp were progressively cooled from 30 degrees C to 23, 17 and 10 degrees C, where they were held for up to 5 days. Endoplasmic reticulum phospholipids showed substantial changes in fatty acid composition, with linear decreases in the proportion of saturates with temperature over the full range of cooling (11.3 % in phosphatidylcholine and 15.8 % in phosphatidylethanolamine). In the phosphatidyl-ethanolamine fraction, this was linked to increased proportions of monoenes, particularly 20:1(n-9). Modest cooling to 23 degrees C on day 1 induced a 2.5-fold transient increase in delta(9)-desaturase activity without any change in the amount of desaturase protein or transcript. Further cooling to 17 degrees C induced a greater and more sustained increase in desaturase activity, reaching sevenfold on day 5, with a 10- to 20-fold increase in the amount of desaturase transcript. Extreme cooling to 10 degrees C led to a very large, but transient, 40- to 50-fold increase in desaturase transcript amounts, a modest 40-50 % increase in desaturase protein but no further increase in activity over that observed at 17 degrees C. These results distinguish at least three mechanisms involved in cold-induced lipid restructuring; the activation of latent desaturase observed with gentle cooling, the induction of desaturase gene transcription and, finally, a third unidentified lipid compensatory mechanism that occurs with extreme cooling. The complex nature of cold-induced lipid restructuring also involves changes in the activity of other biosynthetic enzymes, including elongase and positional- and phospholipid-specific acyltransferases.

Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.

Deletion of the 389 N-terminal residues of the transcriptional activator AREA does not result in nitrogen metabolite derepression in Aspergillus nidulans.

Utilizing a homologous gene replacement in order to retain the native promoter and 5' and 3' untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression in Aspergillus nidulans. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649-6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.

An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Evolution of transcription-regulating proteins: caveat lector!

The paper of Hawkins et al. [Gene 146 (1994) 145-158] reports incorrect descriptions of mutant phenotypes, omits mention of the absence of a highly relevant glutamine-binding site and contains sequence alignments which might mislead the reader. Extensive sequence analysis reveals as untenable a central hypothesis of the paper concerning a possible evolutionary relationship between anthranilate synthetases and the transcription factors mediating nitrogen metabolite repression in fungi.

Genetic and molecular characterization of murine GATA-1 in Aspergillus defines a critical role for the N-terminal finger.

We have utilized Aspergillus nidulans as a model system for the characterization of the major vertebrate transcription factor GATA-1. This has been achieved both by analysing the function of murine GATA-1 directly and by using direct gene replacement to introduce chimaeric areA::GATA-1 derivatives at the areA locus, which encodes a GATA factor involved in regulating nitrogen metabolism in A. nidulans. Although GATA-1 shows only limited function when expressed in A. nidulans, the C-terminal GATA DNA-binding domain can replace the native GATA domain of AREA and retain near wild-type function. Surprisingly, inclusion of the N-terminal DNA-binding domain of GATA-1 has a major role in determining the function of areA::GATA constructs in vivo, leading to a general loss of activation. This negative function is partially dominant and is dependent on both the fidelity of the zinc-chelating structure and a second factor encoded by A. nidulans. The presence of two GATA domains also disrupts modulation of AREA activity. The ability of duplicate GATA domains to disrupt normal signal transduction is not dependent on the relative position of the domains or on the fidelity of the zinc-chelating structure. This demonstrates the utility of nitrogen metabolism's regulation in A. nidulans as a model system for the molecular and genetic characterization of heterologous GATA factors while also providing insights into native Aspergillus regulatory components.

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.

Molecular characterisation of meaB, a novel gene affecting nitrogen metabolite repression in Aspergillus nidulans.

Mutations within the meaB gene elicit the inappropriate expression of several activities subject to nitrogen metabolite repression in Aspergillus nidulans and also have some unrelated phenotypic effects. We have cloned meaB and isolated a full length cDNA clone. Northern analysis has shown that meaB expression is not subject to nitrogen metabolite repression. meaB encodes a novel protein of 418 amino acids and contains a significantly high number of S/TPXX motifs, a motif common in transcriptional regulatory proteins. We have sequenced three mutations within meaB, two of which have an identical phenotype to that produced by gene disruption.

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans.

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

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.

Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans.

In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions.

Nitrogen regulation in fungi.

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems in Aspergillus nidulans and Neurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation of areA, the principle regulatory gene from A. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities in A. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by the areA product, in particular localisation of the domain within the areA product which mediates the regulatory response within the protein.