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C Scazzocchio

Publications and source records attributed to C Scazzocchio.

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

The integration of nitrogen and carbon catabolite repression in Aspergillus nidulans requires the GATA factor AreA and an additional positive-acting element, ADA.

The expression of the structural genes of the proline utilization cluster of Aspergillus nidulans is repressed efficiently only when both repressing carbon and nitrogen sources are present. Two hypotheses can account for this fact. One is a direct or indirect competition mechanism between the positive-acting AreA GATA factor, mediating nitrogen metabolite repression, and the negative-acting CreA protein, mediating carbon catabolite repression. The second is to propose that CreA prevents the binding or activity of another, as yet unidentified, positive-acting factor, here called ADA. We show the second possibility to be the correct one, and we localize the new positive cis-acting element within 290 bp of the prnD-prnB divergent promoter.

Aspergillus nidulans↗
PubMed1997

The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation.

In Aspergillus nidulans a highly specific L-proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism. In mycelia, the expression of the prn structural genes is finely co-regulated in response to proline induction and nitrogen/carbon catabolite repression. In this study we establish that prnB expression is also activated during germination of conidiospores. This activation persists until the development of 6 h-old mycelia and it is independent of proline induction mediated by the pathway-specific prnA gene product. We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation. This process has two components: a prnA-dependent and a prnA-independent component. A cis-acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation. We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium. This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.

Amino Acid Transport Systems, Neutral↗
PubMed1997

Mutations in a dispensable region of the UaY transcription factor of Aspergillus nidulans differentially affect the expression of structural genes.

The uaY gene encodes a transcriptional activator mediating uric acid induction of at least nine genes of the purine-utilization pathway. In this article, we characterize a loss-of-function mutation, uaY205, as a 16 bp deletion that results in premature translation termination, and substitutes the C-terminal 63 amino acids for 13 amino acid residues. Reversion analysis demonstrates that the C-terminal 63 amino acid residues are unnecessary for UaY function, and that the loss-of-function phenotype resulting from the uaY205 mutation is caused by the new amino acid sequence present in the mutant protein. Revertants in two different frames (wild type and +1) restore function but show subtle differences in the expression of genes controlled by the UaY protein. Two strains showing elevated expression of genes under UaY control were shown to carry, in addition to a mutation leading to the recovery of the wild-type open reading frame, mutations in unlinked genes. Using crude extracts of Aspergillus nidulans, we have been able to detect, for the first time, in transcription factors of this class, specific retardation of a promoter probe. The binding activity is at least partially dependent on the presence of inducer. The gel shift experiments show that the novel inhibitory sequence present in the UaY205 protein can act either by affecting the stability of the protein, or via an inter- or intramolecular interaction impairing the specific DNA-binding activity.

Amino Acid Sequence↗
PubMed1995

Post-transcriptional control and kinetic characterization of proline transport in germinating conidiospores of Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, L-proline uptake is mediated by the product of the prnB gene which codes for a member of a family of amino acid transporters found both in pro- and eukaryotes. Regulation of prnB gene expression has previously been studied in great detail at the molecular level. However, no studies have addressed possible post-transcriptional controls or the kinetic characterisation of the PrnB transporter. Here we develop a rapid and efficient method for direct uptake measurements of proline in germinating conidiospores of A. nidulans. We make use of this method and Northern blot analyses in parallel to study the regulation of PrnB expression both at the level of prnB message accumulation and at a post-transcriptional level. These studies show that (i) pathway-specific and wide-domain regulatory systems, previously shown to control prnB gene expression in multicellular mycelia, also operate in unicellular conidia committed to germination; and (ii) PrnB activity is regulated in response to the nitrogen source present in the medium and the level of internally accumulated proline or other amino acids. We also characterise kinetically the PrnB transporter and a secondary proline transport system. Our results open new possibilities for studies using unicellular conidiospores of filamentous fungi and constitute a necessary first step for a subsequent structure-function analysis of the PrnB transporter.

Amino Acids↗
PubMed1995

A single amino acid change in a pathway-specific transcription factor results in differing degrees of constitutivity, hyperinducibility and derepression of several structural genes.

unYc462 is a gain-of-function mutation in the purine catabolism positive regulatory gene of Aspergillus nidulans. This allele leads to a constitutive, hyperinducible and derepressed expression of a least three genes controlled by uaY, and this occurs at different levels depending on the target gene. The uaYc462 allele was mapped physically in relation to known loss-of-function alleles and sequenced. uaYc462 is a one-base change in codon 222, resulting in a serine to leucine change. We propose that this mutation maps in a functional domain involved, directly or indirectly, in the interaction of UaY with other components of the transcriptional apparatus. A sequence similar to the motif surrounding serine 222 may play similar roles in the PPR1 and ADR1 proteins of Saccharomyces cerevisiae.

Amino Acid Sequence↗
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 sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae.

The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans. Here we present the complete genomic and cDNA nucleotide sequence of this gene. The gene contains two introns. The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae. These similarities are most striking in the putative linker and dimerization regions following the zinc cluster. Gel-shift and DNase I footprinting experiments have been carried out for three genes subject to UaY-mediated induction. The binding sequence is 5'-TCGG-6X-CCGA, which is identical to the proposed PPR1 binding sites. Nevertheless, the identity of the base immediately 3' of the 5'-TCGG sequence clearly affects the affinity of the site. The site upstream of the uapA gene has been shown to be active in vivo. Binding to this site has been analysed by a number of interference techniques. There is an interesting chemical similarity between the co-inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control. These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S. cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY.

Amino Acid Sequence↗
PubMed1995

Cloning and molecular characterization of hxA, the gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans.

We have cloned and sequenced the hxA gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. The gene codes for a polypeptide of 1363 amino acids. The sequencing of a nonsense mutation, hxA5, proves formally that the clones isolated correspond to the hxA gene. The gene sequence is interrupted by three introns. Similarity searches reveal two iron-sulfur centers and a NAD/FAD-binding domain and have enabled a consensus sequence to be determined for the molybdenum cofactor-binding domain. The A. nidulans sequence is a useful outclass for the other known sequences, which are all from metazoans. In particular, it gives added significance to the missense mutations sequenced in Drosophila melanogaster and leads to the conclusion that while one of the recently sequenced human genes codes for a xanthine dehydrogenase, the other one must code for a different molybdenum-containing hydroxylase, possibly an aldehyde oxidase. The transcription of the hxA gene is induced by the uric acid analogue 2-thiouric acid and repressed by ammonium. Induction necessitates the product of the uaY regulatory gene.

Amino Acid Sequence↗
PubMed1995

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

Aspergillus nidulans↗
PubMed1994

Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans.

CREA is the negative regulator mediating carbon catabolism repression in Aspergillus nidulans. We have determined all the sites in the DNA region between the prnD and prnB genes of the proline degradation cluster of this organism which are able to bind a fusion protein containing the zinc finger domain of CREA. The consensus sequence derived for CREA binding is 5'-SYGGRG-3', but not all possible sites derived from this consensus do in fact bind. The binding of at least some sequences of the form 5'-SYGGAG-3' is context dependent. Two different and divergent sites, separated by one base pair (5'-GCGGAGACCCCAG-3'), contain the previously sequenced derepressed mutations and are essential for carbon catabolite repression in vivo. We have studied the binding of CREA to the region by DNase I and methylation protection, and by methylation and depurination interference. We propose that this pair of sites is the physiological, cis-acting element responsible for the carbon catabolite repression of prnB transcription.

Aspergillus nidulans↗
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↗
PubMed1993

Sequence and regulation of the uapA gene encoding a uric acid-xanthine permease in the fungus Aspergillus nidulans.

The nucleotide sequence of the uapA gene, coding for the uric acid-xanthine permease of Aspergillus nidulans, has been determined. The predicted uapA gene product comprises 595 amino acids (M(r) 63,365); it is a highly hydrophobic protein with 12-14 putative transmembrane segments and shows no striking similarity to any other membrane protein of either prokaryotes or eukaryotes, except for a short highly hydrophobic amino acid sequence conserved in a number of different permeases. The presence of an acidic, amphipathic region overlapping with the last hydrophobic segment of UAPA could also be of interest. The results presented suggest that the UAPA permease represents a new type of membrane protein, not described previously. The transcription of uapA is inducible by 2-thiouric acid, and it is highly repressible by ammonium. It is almost absolutely dependent on the presence of functional uaY and areA regulatory gene products. A specific mutation in the GATA binding zinc finger of the AREA protein nearly abolishes uapA transcription. The uap100 cis-acting, up-promoter, constitutive mutation is a duplication that comprises two GATA sites and suppresses weakly the AREA zinc finger mutation but does not alleviate the need for functional UAY and AREA proteins.

Amino Acid Sequence↗