Production and characterization of polyclonal antibodies against norsolorinic acid reductase involved in aflatoxin biosynthesis

Polyclonal antibodies against norsolorinic acid reductase (NSR), an enzyme responsible for the conversion of norsolorinic acid to averantin in the early stage of aflatoxin biosynthesis, were produced after immunizing rabbits with a semi‐purified NSR preparation. Immunochemical analysis of the enzyme extracts from Aspergillus parasiticus revealed that the antibodies reacted with several protein species, including bands corresponding to NSR activity. The ammonium sulfate‐cut IgG was further purified by passing it through a column armed with protein fractions containing immunoreactivity, but no enzyme activity, isolated from the A. parasiticus extracts that had been subjected to Sepharose gel filtration. After subtractive affinity chromatography, the purified antiserum reacted primarily with two protein bands (of molecular weight 43 and 48 kDa) and was capable of neutralizing the NSR activity. Enzyme‐linked immunosorbent assay (ELISA) analysis of various fungal extracts showed that the purified antiserum was highly specific for the enzyme.     

Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5^- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.     

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans

Summary Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.Deceased April 30, 1988     

Ammonium ion sensitivity is a ribosomal phenotype associated with suppressor mutations in the suaC gene of Aspergillus nidulans

Summary Ammonium ions are selectively toxic to strains containing mutations in the suaC gene which can mutate to a suppressor phenotype. This phenotype is associated with increased ribosomal misreading in vitro (Zamir and Martinelli 1987) and altered ribosomal proteins (Harvey and Martinelli 1983). Such ammonium-sensitivity is a feature of both strong and weak suppressor alleles, and segregates with suppressor ability in crosses. Suppressor mutations in the suaB and suaD genes are not affected, nor are those in suaA, another ribosomal suppressor gene. Thus, the ammonium-effect is locus specific. Mutations which act as antisuppressors (asu ^–) of suppressor suaC109 also partially reverse the ammonium ion sensitivity associated with this mutation. This effect is in line with their restoration of other aspects of the pleiotropic phenotype to normal. The cations, lithium and rubidium, mimick the effects of ammonium ions. Only ribosomes from suaC strains are sensitive to the presence of NH ₄ ⁺ ions in vitro.     

Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae

To investigate the structure of the aflatoxin gene cluster in Aspergillus oryzae, 39 strains belonging to this species were examined for the existence of pksA, fas-1A, aflR and vbs, and the results compared with those for ver-1 obtained previously. These five genes are involved in aflatoxin biosynthesis in Aspergillus parasiticus. The strains examined were categorized into three groups; group 1, having the five homologs; 2, having ver-1 and vbs; and 3, having vbs homologs. Long-PCR analysis of the regions between the five homologs in A. oryzae IFO 4135, coupled with Southern-hybridization analysis, shows that those homologs are clustered with the same arrangement as in A. parasiticus. These results suggest that directed deletions of the cluster occur in A. oryzae strains. The possible breakpoints of the deletions in the strains of group 2 and 3 were estimated. Received: 23 April / 8 November 1999     

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): Indications of a pectin lyase gene family in A. niger

Summary Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the loboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commeriial pectinase preparation Ultrazym^®. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5 or 3 parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym^®, PLII.     

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

Investigation of the action of aflatoxin B1 in vitro andin vivo

The direct and transplacental action of aflatoxin B₁ was compared in organ cultures of embryonic lung tissue of strain A mice, BD-IX rats, and golden hamsters. It was shown to have a toxic action on the culture but no carcinogenic effect. In experiments on mice transplacental penetration of aflatoxin B₁ led to an increased frequency of mammary gland tumors only in the progeny.Academician of the Academy of Medical Sciences of the USSR.Department for the Study of Carcinogenic Agents, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1349–1352, November, 1976.     

Detoxication of benzene and aflatoxin B1 with amino acid and peptide preparations in mice and chicken

Incubation of mouse and chicken splenocytes with amino acid or peptide preparationsin vitro increases cell resistance to benzene and aflatoxin B₁. Short-term (15 days) treatment of chicken with an amino acid mixture (aviamine) in combination with benzene also increased splenocyte resistance to toxinin vitro. By contrast, aviamine in combination with aflatoxin B₁ sharply decreased cell resistance to toxin. Glutamic acid possessed no such properties. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 419–421, October, 1999     

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

Summary Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T₂ gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T₂ revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T₂ activity than wild-type strains.     

Aspergillus fumigatus infection in a green parrot

Aspergillosis is a common mycotic disease of parrots which occurs as an acute infection of young parrots. The dehydrated and emaciated carcass of a 1-year-old green parrot was referred for post-mortem examination. On necropsy, disseminated variably sized granulomas were seen. At microscopic examination, they were found to contain intra-lesional septate and branching fungal hyphae. Lesions were extensive around the thoracic inlet and abdominal cavity. Aspergillus fumigatus was cultured from the visceral and thoracic granulomas. The source of exposure to the microorganism was not determined.     

Chromosomal mapping and gene disruption of the ADHIII gene in Aspergillus nidulans

Summary There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHIII has no obvious physiological function. We describe here the cloning of the ADHIII gene (alcC), its mapping on linkage group VII by reverse genetics, and the properties of multicopy transformants tested for their ability to grow on a range of alcohols (butan-1-ol being the best substrate tested for growth). We were unable to detect any obvious alteration in phenotype of a strain carrying a disrupted copy of the ADHIII gene.     

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

Summary Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.The sequence reported in this paper has been submitted to the EMBL/GenBank through DDBJ (accession no, X52525).     

Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its expression in Saccharomyces cerevisiae

The cDNA encoding the endo--1,4-glucanase (carboxymethylcellulase; CMCase-I) from Aspergillus kawachii IFO 4308 was cloned. Nucleotide-sequence analysis of the cloned cDNA insert showed a 717-bp open reading frame that encoded a protein of 239 amino-acid residues. The predicted amino-acid sequence of the mature protein had considerable homology with the protein sequence of the FI-CMCase of Aspergillus aculeatus. The cDNA was introduced into Saccharomyces cerevisiae. The expressed enzyme had carboxylmethylcellulase acitivity, identified by clear zones on a CMC-agar plate after Congo Red staining.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants

Summary The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.     

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library

Autonomously-replicating, marker-less helper plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.     

The oliC3 gene of Aspergillus niger: isolation,sequence and use as a selectable marker for transformation

Summary The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycinresistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5 untranslated sequences with those from other fungi.     

Suppressor specificity in Aspergillus nidulans

Summary Seven allele specific gene unspecific suppressors mapping at four loci have been described previously (Roberts et al. 1979). Three new suppressors mapping in suaA are characterised, and the spectrum of suppression of all the suppressors with respect to seventeen suppressible mutations in eight different genes is described. Two distinct classes of suppressor are defined. The diversity of suppression of five suaA alleles, and the temperature sensitivity of some suaA suppressor mutant combinations but not others, suggests that suppressors at this locus are acting via ribosomal protein alteration. suaC109, a mutation that results in cold-sensitivity for growth shows a similar broad spectrum of suppression. Suppressors at the suaA and suaC loci suppress mutations that have the properties of chain termination mutations as well as missense mutations. suaB111, and suaD103 and suaD108 have a very restricted range of suppression. These suppressors may be mutations in tRNA genes.     

Mutations to constitutivity and derepression are separate and separable in a regulatory gene of Aspergillus nidulans

Summary Previous work has shown that expression of the structural genes for the enzymes of nitrate and nitrite assimilation in Aspergillus nidulans requires the products of two positively acting regulatory genes — nirA, mediating induction, and areA, mediating nitrogen metabolite repression. Here we show that, in addition to previously described mutations in nirA leading to constitutivity, other mutations can be selected in nirA leading to nitrogen metabolite derepression. These constitutivity and depression mutations in nirA are additive and separable by intragenic recombination. This suggests that the nirA gene product contains two separate domains, a co-inducer binding region, defined by constitutivity mutations, and a region interacting with the areA gene product or with initiator sites adjacent to structural genes under areA and nirA control, defined by derepression mutations. These findings might indicate a striking similarity of action between the eukaryotic regulatory gene nirA and a comparable prokaryotic regulatory gene.