Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans.

The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus Aspergillus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASalc) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.

Magnetocardiography measurements with 4He vector optically pumped magnetometers at room temperature.

In this paper, we present a proof of concept study which demonstrates for the first time the possibility of recording magnetocardiography (MCG) signals with 4He vector optically pumped magnetometers (OPM) operated in a gradiometer mode. Resulting from a compromise between sensitivity, size and operability in a clinical environment, the developed magnetometers are based on the parametric resonance of helium in a zero magnetic field. Sensors are operated at room temperature and provide a tri-axis vector measurement of the magnetic field. Measured sensitivity is around 210 f T (√Hz)-1 in the bandwidth (2 Hz; 300 Hz). MCG signals from a phantom and two healthy subjects are successfully recorded. Human MCG data obtained with the OPMs are compared to reference electrocardiogram recordings: similar heart rates, shapes of the main patterns of the cardiac cycle (P/T waves, QRS complex) and QRS widths are obtained with both techniques.

First experimental evidence of a zinc binuclear cluster in AlcR protein, mutational and X-ray absorption studies.

AlcR is the transcriptional activator of the ethanol utilization pathway in Aspergillus nidulans. The zinc DNA-binding domain contains ligands of zinc, six cysteines (Zn2Cys6) or five cysteines and one histidine (Zn2Cys5His). The utilisation of complementary approaches such as X-ray absorption spectroscopy, mutational analysis, zinc content evaluation, determination of specific binding connecting structural and biological data, have allowed to determine zinc environment and to analyse the involvement of amino acids. The determination by EXAFS of zinc ligands (four sulphur atoms), the Zn content in the protein (2:1), the evaluation of the distance between two zinc atoms (3.16 +/- 0.02 angstroms), together with the total loss of specific DNA-binding activity when one cysteine ligand is mutated, are in favour of a zinc cluster model in which six cysteine sulphurs ligate two zinc atoms. XANES spectra of wild type and H10A AlcR protein are virtually identical indicating that Histidine 10 does not have a direct contribution in zinc ligation but electrophoretic mobility shift assays show that His10 is involved in DNA-binding. In contrast, proline 25 does not seem to play any direct role in the DNA-binding activity but XANES spectra of Pro25A AlcR protein are slightly modified comparing to the wild type protein spectra. This suggests a role of the proline in the stabilisation of the Zn cluster structure. AlcR DNA-binding domain belongs to the zinc binuclear class family (Zn2Cys6) with unique characteristics resulting from its primary and secondary structures and its binding specificity toward direct and inverted repeat target.

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half-site DNA: a NMR study.

The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half-site 5'-TGCGG-3'. Apparent microM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74+/-7 ms at 20 degrees C with the following DNA sequence: 5'-C1G2T3G4C5G6G7A8T9C10-3'. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half-site is another example of the variability in contacts of the fungal DNA-binding proteins containing Zn2Cys6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.

In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class.

AlcR is the transactivator mediating transcriptional induction of the alc gene cluster in Aspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn2Cys6 family by several features. In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A. nidulans. Here, we show that unlike the other proteins of the Zn2Cys6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets. Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats. The consensus core has been determined precisely (5'-CCGCN-3'), and the AlcR-binding site in the aldA promoter has been localized. The sequence downstream of the zinc cluster is necessary for high affinity binding. Furthermore, our data show that the use of the carrier protein glutathione S-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure.

Unique DNA binding specificity of the binuclear zinc AlcR activator of the ethanol utilization pathway in Aspergillus nidulans.

AlcR is the transcriptional activator in Aspergillus nidulans, necessary for the induction of the alc gene cluster. It belongs to the Zn2Cys6 zinc cluster protein family, but contains some striking differences compared with other proteins of this group. In this report, we show that no dimerization element is present in the entire AlcR protein which occurs in solution as a monomer and binds also to its cognate sites as a monomer. Another important feature of AlcR is its unique specificity for single sites occurring naturally as inverted or direct repeats and sharing a common motif, 5'-(T/A)GCGG-3'. Like most other Zn2Cys6 proteins, AlcR contacts directly with the CGG triplet and, in addition, the upstream adjacent guanine is required for high affinity binding. We also establish that the flanking regions outside the core play an essential role in tight binding. From our in vitro analysis, we propose an optimal AlcR-binding site which is 5'-PuNGCGG-AT rich 3'.

A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus nidulans.

In Aspergillus nidulans, the transcriptional activator AlcR mediates specific induction of a number of alc genes. The AlcR DNA-binding domain is a zinc binuclear cluster that differs from the other members of the Zn2Cys6 family in several respects. Of these, the most remarkable is its ability to bind in vitro as a monomer to single sites, whereas only repeated sites (direct or inverted) are necessary and functional in vivo. Deletion of the first five amino acids (following the N-terminal methionine) upstream of the AlcR zinc cluster or mutation of a single residue, Arg-6, impairs the AlcR in vitro binding mainly to symmetrical sites. In vivo, the same mutations result in the inability of A. nidulans to grow on ethanol. The alc- phenotype results from a drastic decrease in activation of its own transcription and, in addition, that of the two structural genes, alcA and aldA, required for ethanol oxidation. This defect seems to be correlated to the inability of the Arg-6 AlcR mutant protein to bind to AlcR palindrome targets, which are essential in the three alc promoters. AlcR shows a unique pattern of binding and of transactivation among the Zn2Cys6 family.

Universal signals control slime mold stalk formation.

The primitive slime mold Dictyostelium minutum does not display oscillations during aggregation, cannot form migrating slugs, and does not form a prestalk/prespore pattern, all of which are characteristic for development of its advanced relative Dictyostelium discoideum. We used D. minutum to investigate whether slime molds share common mechanisms controlling development. In D. discoideum, the morphogen differentiation inducing factor (DIF) can induce stalk-cell differentiation in vitro. However, stalk formation in vivo is supposedly triggered by local depletion of DIF antagonists such as ammonia or cAMP. A homologue of the D. discoideum stalk gene ecmB was cloned in D. minutum that encodes a 3.4-kb mRNA, and its deduced amino acid sequence shows repeats of 24 amino acids that are characteristic for the D. discoideum ecmB gene. Remarkably, DIF effectively induces expression of the D. minutum ecmB gene and ammonia inhibits its expression. D. discoideum cells were transformed with a construct of the D. minutum ecmB promoter fused to the lacZ reporter gene and showed expression in the stalk, but not in the upper and lower cup of the fruiting body, which also express the D. discoideum ecmB gene. In D. discoideum, the D. minutum ecmB and the ecmB promoter are similarly activated by DIF and repressed by both cAMP and ammonia, suggesting that additional signaling is required for ecmB expression in upper and lower cup cells. Our data indicate that the extracellular signals controlling stalk formation and their intracellular signaling cascades including gene regulatory proteins remained highly conserved during slime mold evolution.

Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans.

ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase. ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster. This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli. Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter. DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3'. The half-sites are separated by a variable number of nucleotides in both types of target. The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays. In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not. Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites. The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion. Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.

Isolation of UV-induced mutations in the areA nitrogen regulatory gene of Aspergillus niger, and construction of a disruption mutant.

The areA gene of filamentous fungi encodes a positive-acting transcriptional factor required for the expression of genes involved in the utilisation of nitrogen sources other than ammonium and glutamine. In Aspergillus niger we have isolated three UV-induced areA mutants and constructed a well-defined disruption allele of the areA gene. The areA gene was genetically localised on Linkage Group III, 3.6 map units (m.u.) from bioA1 and 4.5 m.u. from lysA7. Analysis of the expression of the nitrate reductase encoding gene and of nitrate reductase activities show that the mutated areA strains behave as loss-of-function mutants and can be classified as areAr type. In addition, growth tests were performed using several nitrogen sources in combination with glucose. The results suggest that, unlike the case in A. nidulans, in A. niger the AreA protein also plays a role in the presence of ammonium. Furthermore, the spectrum of protease activities secreted by A. niger differs from that produced by A. nidulans, as only A. niger is able to degrade elastin.