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C Scazzocchio

Publications and source records attributed to C Scazzocchio.

At least 55 records · Page 3Linked to original sources
PubMed1993

Sequence, regulation, and mutational analysis of the gene encoding urate oxidase in Aspergillus nidulans.

The uaZ gene of Aspergillus nidulans codes for a protein of 301 amino acids. The open reading frame is interrupted by two introns. A comparison with the open reading frames of 10 other urate oxidases reveals a number of scattered universally conserved residues and four clusters of high similarity. Two of these are of unknown function, and the other two are the putative sites for copper binding and the signal sequence for peroxisomal entry, respectively. This comparison also reveals that there are at 16 positions at which introns can be found in the gene coding for this enzyme in different species. The sequence of a number of mutations confirms the importance for enzyme activity of the carboxyl terminus of the protein. The most amino-terminal mutation (an ochre codon) results also in strikingly diminished steady-state levels of message. This agrees with results from other systems, suggesting that untranslated mRNA is preferentially degraded in eukaryotic cells. Northern blots show that specific induction, mediated by the UAY protein, and nitrogen metabolite repression, mediated by the AREA GATA factor, affect the steady-state levels of uaZ mRNA and thus act most probably at the level of transcription.

Alleles↗
PubMed1993

Characterisation, cloning and integrative properties of the gene encoding urate oxidase in Aspergillus nidulans.

A number of mutations have been obtained which define the structural gene (uaZ) coding for urate oxidase in linkage group I of Aspergillus nidulans. This gene has been cloned by transformation of a uaZ- null mutant. A chromosome I/VIII translocation which splits the gene has been defined both genetically and physically. All known mutations are contained in a 1-kb fragment, itself contained in the probe which recognizes a 1.2-kb inducible message. Plasmids carrying uaZ show a strict bias towards homologous recombination in transformation experiments.

Alleles↗
PubMed1993

Operator derepressed mutations in the proline utilisation gene cluster of Aspergillus nidulans.

The proline utilisation gene cluster of Aspergillus nidulans can be repressed efficiently only when both repressing nitrogen and repressing carbon sources are present. We show that two cis-acting mutations in this cluster permit the efficient transcription of the prnB gene under repressing conditions, resulting in direct or indirect derepression of two other transcripts of the pathway. These mutations are transitions that define a 5'GAGACCCC3' sequence. Similar sequences are found upstream of other genes subject to carbon catabolite repression. We propose that this sequence defines the binding site for the negatively-acting CreA protein, which mediates carbon catabolite repression in this fungus.

Amino Acid Sequence↗
PubMed1991

Heterologous expression of the Aspergillus nidulans regulatory gene nirA in Fusarium oxysporum.

We have isolated strains of Fusarium oxysporum carrying mutations conferring a phenotype characteristic of a loss of function in the regulatory gene of nitrate assimilation (nirA in Aspergillus nidulans, nit-4 in Neurospora crassa). One of these nir- mutants was successfully transformed with a plasmid containing the nirA gene of A. nidulans. The nitrate reductase of the transformants is still inducible, although the maximum activity is lower than in the wild type. Single and multiple integration events were found, as well as a strict correlation between the presence of the nirA gene and the Nir+ phenotype of the F. oxysporum transformants. We also investigated how the A. nidulans structural gene (niaD) is regulated in F. oxysporum. Enzyme assays and Northern experiments show that the niaD gene is subject to nitrate induction and that it responds to nitrogen metabolite repression in a F. oxysporum genetic background. This indicates that both the mechanisms of specific induction, mediated by a gene product isofunctional to nirA, and nitrogen metabolite repression, presumably mediated by a gene product isofunctional to the homologous gene of A. nidulans, are operative in F. oxysporum.

Aspergillus nidulans↗
PubMed1991

Correct intron splicing generates a new type of a putative zinc-binding domain in a transcriptional activator of Aspergillus nidulans.

alcR is the pathway-specific transcriptional activator of the ethanol regulon in the filamentous fungus, Aspergillus nidulans. The deduced amino acid sequence of a cDNA clone, including the 5' part of the alcR-mRNA, shows that a putative Zn-binding domain of the all-cysteine class, exemplified by GAL4 is present. This structure presents some striking features. At variance with other structures of this class, the binding domain is strongly asymmetrical. Model building indicates that the zinc-binding motif of alcR could adopt an helix-turn-helix structure. We propose that the DNA-binding motif of alcR could participate in two types of DNA-binding structures: the zinc-cluster and the helix-turn-helix.

Amino Acid Sequence↗
PubMed1991

The uaY positive control gene of Aspergillus nidulans: fine structure, isolation of constitutive mutants and reversion patterns.

The product of the uaY gene of Aspergillus nidulans is necessary for the expression of at least eight genes coding for enzymes and permeases of the purine utilisation pathway. A detailed fine structure map has been constructed of this gene involving 13 presumed point mutations and eight deletions. Gene conversion of these deletions was demonstrated. A technique was devised to select for constitutive mutations and two were obtained which map within the uaY gene. We have shown that the most centromere proximal allele reverts to a number of different phenotypes. The properties of this allele suggest that it may map in the open reading frame of the uaY gene, in a domain that could be altered in a way that would differentially affect the expression of genes under uaY control.

Aspergillus nidulans↗
PubMed1991

Molecular cloning of the uaY regulatory gene of Aspergillus nidulans reveals a favoured region for DNA insertions.

The cloning of the positive regulatory gene, uaY, which mediates uric acid induction of enzymes and permeases of the purine degradation pathway in the fungus Aspergillus nidulans is described here. The 4 kb uaY transcript is constitutively synthesised, it is not repressed by ammonia and its transcription does not require the AreA wide-domain transcription factor. We have determined that four deletions, which have been genetically characterised, are confined to a segment of 0.9 kb. Two other deletions are double events; each is a deletion of about 1 kb plus an insertion. The positions of the deletions confine 9 out of the 11 mapped putative point mutations within a 1 kb segment. Two other non-revertible alleles, which mapped as point mutations, are insertions of at least 11 and 18 kb respectively. The pattern of gene conversion within the uaY gene was described previously. The results reported here demonstrate that conversion of sequences of at least 18 kb can occur in A. nidulans.

Alleles↗
PubMed1991

nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions.

The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans.

We have cloned an 11-kbp segment of the genomic DNA of Aspergillus nidulans which complements mutations in nirA, the pathway-specific regulatory gene of the nitrate assimilation pathway. Gene disruption in the corresponding region of the nuclear DNA leads to a phenotype and a gene complementation pattern indistinguishable from that observed in known noninducible nirA mutants. Transformation studies with subclones of the 11-kbp genomic segment showed that a nonreverting null mutation nirA87, maps to a 1.5-kbp stretch within that segment. These data confirm that the cloned segment contains the nirA gene. The gene is completely encompassed in the 11-kbp genomic segment, as a plasmid carrying the corresponding insert gives rise to multicopy transformants exhibiting better growth than wild type on nitrate or nitrite as the sole nitrogen source. Southern and genetic analyses of transformants obtained with various plasmid subclones established a gene size of at most 5.9 kbp. Northern (RNA) hybridization experiments revealed a 4-kb nirA transcript which is barely visible in the wild type but clearly seen in a transformant carrying about 10 gene copies. In both strains, nirA mRNA is synthesized constitutively. Upstream of nirA, a neighboring transcript about 2.8 kbp in length which is transcribed from the opposite strand with respect to nirA was localized. The transcript levels of niaD and niiA, encoding the nitrate and nitrite reductase core proteins, respectively, were investigated in nirA mutants and a nirA multicopy transformant. The results show that the nirA product regulates the transcript steady-state level of these structural genes and that it is a limiting factor for their expression.

Aspergillus nidulans↗
PubMed1990

Insertional inactivation and cloning of the wA gene of Aspergillus nidulans.

We describe examples of wA gene inactivation (resulting in white conidiospores) obtained during transformation of Aspergillus nidulans. One wA- transformant was obtained by transformation with a prn+ plasmid of a strain with green conidia (wA+) which was unable to catabolize L-proline (prn-). This transformant contains a very large number of plasmid copies integrated at a single site inseparable from the wA locus. Passage of this transformant through the sexual cycle generated a variety of novel phenotypes for L-proline utilization, the number and frequency of which depended upon the cleistothecium from which the progeny were obtained, suggesting that the altered phenotypes were due to premeiotic events. The most extreme phenotype was severe hypersensitivity to L-proline. Hypersensitive progeny had a much reduced number of integrated plasmid copies enabling us to identify and clone putative prn-wA fusion sequences and subsequently retrieve wA sequences from a wild-type gene library. One of the wild-type clones overlapped the different sites of the insertional mutations in two wA- transformants and complemented the wA3 allele. Sequences within this clone hybridized to a transcript that was developmentally regulated in the wild type and absent in a number of mutants defective in conidiospore development. A reiterated sequence was also found in the region of the wA gene.

Aspergillus nidulans↗
PubMed1989

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

Adenosine Triphosphatases↗
PubMed1989

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum.

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.

Aspergillus nidulans↗
PubMed1989

A gene coding for the uric acid-xanthine permease of Aspergillus nidulans: inactivational cloning, characterization, and sequence of a cis-acting mutation.

In Aspergillus nidulans, integration of transforming sequences can proceed through recombination with homologous sequences or at heterologous sites in the genome. In a strain with a large deletion in the gene coding for acetamidase (amdS), a plasmid carrying this gene integrates into and inactivates uapA, the putative structural gene for uric acid-xanthine permease, with a frequency of 0.3%. The integration event occurs 3' to the open reading frame of amdS. A 10-nucleotide sequence which occurs in this region is also found within the open reading frame of uapA. We have taken advantage of this integration event to clone the permease gene and to characterize a cis-acting mutation, uap-100, as a duplication of 139 bp located in the upstream region of uapA. Northern and dot blot analyses confirmed earlier results measuring the uptake of uric acid: the transcription of the uapA gene is inducible and the uap-100 mutation results in a bypass of the need for induction while having an 8-fold up-promoter effect under inducing conditions.

Aspergillus nidulans↗