Isolation and characterisation of the acetyl-CoA carboxylase gene from Aspergillus nidulans

The putative gene encoding acetyl-CoA carboxylase, accA, has been isolated from Aspergillus nidulans. This single-copy gene has an open reading frame (ORF) of 6864 bp and contains two small introns near the 5′-end. A short ORF upstream of the ATG start codon has been identified in this gene by RT-PCR. Based on sequence homology to acetyl-CoA carboxylases from other organisms, putative biotin-, ATP-, HCO₃ ^–- and acetyl-CoA- binding sites have been assigned. Northern data and ACC enzyme-activity measurements from A. nidulans suggested that expression of accA was higher in media containing nitrate than ammonia as a sole nitrogen source. Deletion of accA in A. nidulans was unsuccessful. The failure of A. nidulans to grow in the presence of the ACC-specific inhibitor, soraphen A, supplemented with C_16–18 fatty acids suggested that ACC is an essential enzyme. Received: 29 January / 26 September 1998     

Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE1

In filamentous ascomycetes, glucose repression is mediated by CRE1, a zinc-finger protein related to Mig1p from yeast. Five putative AMPK phosphorylation motifs identified in the glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum were mutated in a GFP::CRE1 translational fusion. Complementation experiments in Aspergillus nidulans and fluorescence microscopy analyses showed that mutation of one site (Ser₂₆₆) abolishes the repressor activity of the fusion protein but not its nuclear targeting, suggesting that an AMPK protein kinase may be involved in the function of the fungal glucose repressor. Received: 23 November 1999 / 25 January 2000     

Identification of a new antifungal target site through a dual biochemical and molecular-genetics approach

 The target site of the antifungal compound LY214352 [8-chloro-4-(2-chloro-4-fluorophenoxy) quinoline] has been identified through a dual biochemical and molecular-genetics approach. In the molecular-genetics approach, a cosmid library was prepared from an Aspergillus nidulans mutant that was resistant to LY214352 because of a dominant mutation in a single gene. A single cosmid (6A6-6) that could transform an LY214352-sensitive strain of A. nidulans to LY214352-resistance was isolated from the library by sib-selection. Restriction fragments from cosmid 6A6-6 containing the functional resistance gene were identified by transformation, and sequenced. The LY214352-resistance gene coded for a protein of 520 amino acids that had a 34% identity and a 57% similarity in a 333 amino-acid overlap to E. coli dihydroorotate dehydrogenase (DHO-DH). The results of a series of biochemical mechanism-of-action studies initiated simultaneously with molecular-genetic experiments also suggested that DHO-DH was the target of LY214352. Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I₅₀=40 ng/ml) and a highly resistant mutant (I₅₀>100 μg/ml) conclusively demonstrated that DHO-DH is the target site of LY214352 in A. nidulans. Several mutations in the DHO-DH (pyrE) gene that resulted in resistance to LY214352 were identified.     

A mitochondrial reading frame which may code for a second form of ATPase subunit 9 in Aspergillus nidulans

Summary The nucleotide sequence of a 74 codon reading frame from the Aspergillus nidulans mitochondrial genome is presented. The derived amino acid sequence displays typical features of dicyclohexylcarbodiimide (DCCD) binding proteins and is 84% homologous with a mitochondrial reading frame that potentially encodes an ATPase subunit 9 polypeptide in Neurospora crassa. However, in A. nidulans, as in N. crassa, there is strong biochemical and genetic evidence that this subunit is in fact nuclearly-encoded. In both organisms the DCCD-binding protein found in the F₀ complexes of mitochondria from actively-growing cultures is almost certainly the product of this nuclear gene, and definitely not that of the mitochondrial reading frame. The discovery of an intact open reading frame than can code for a DCCD-binding protein in the mitochondrial genome of a second species of filamentous fungus strenghthens the possibility that the presence of a mitochondrial version of this gene has some biological significance.     

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′′-monophosphate decarboxylase

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5′-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function. Received: 12 December 1997 / 11 March 1998     

Supplementation of Aspergillus glaucus with gfdB gene encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans

In Aspergillus nidulans, there are two putative glycerol 3-phosphate dehydrogenases encoded by the genes gfdA and gfdB, while the genome of the osmophilic Aspergillus glaucus harbors only the ortholog of the A. nidulans gfdA gene. Our aim was to insert the gfdB gene into the genome of A. glaucus, and we reached this goal with the adaptation of the Agrobacterium tumefaciens-mediated transformation method. We tested the growth of the gfdB-complemented A. glaucus strains on a medium containing 2 mol l^‒1 sorbitol in the presence of oxidative stress generating agents such as tert-butyl hydroperoxide, H₂O₂, menadione sodium bisulfite, as well as the cell wall integrity stress-inducing agent Congo Red and the heavy metal stress eliciting CdCl₂. The growth of the complemented strains was significantly higher than that of the wild-type strain on media supplemented with these stress generating agents. The A. nidulans ΔgfdB mutant was also examined under the same conditions and resulted in a considerably lower growth than that of the control strain in all stress exposure experiments. Our results shed light on the fact that the gfdB gene from A. nidulans was also involved in the stress responses of the complemented A. glaucus strains supporting our hypothesis on the antioxidant function of GfdB in the Aspergilli. Nevertheless, the osmotolerant nature of A. glaucus could not be explained by the lack of the gfdB gene in A. glaucus, as we hypothesized earlier.     

Pea chloroplast genes encoding a 4kDa polypeptide of photosystem I and a putative enzyme of C1 metabolism

Summary The nucleotide sequence of 3.2 kbp of pea chloroplast DNA located upstream from the petA gene for cytochrome f, and previously reported to contain the gene for a photosystem I polypeptide, has been determined. Three open reading frames of 587, 40 and 157 codons have been identified. Orf40 encodes a highly conserved, hydrophobic, membrane-spanning polypeptide, and is identified as the gene psaI for the 4 kDa subunit of photosystem I. Orf587 is an extended version of the gene zfpA previously identified as encoding a conserved putative zinc-finger protein. The product of orf587 shows extensive homology to an unidentified open reading frame cotranscribed with a gene for folate metabolism in Escherichia coli and local homology to a region of the subunit of rat mitochondrial propionyl-CoA carboxylase. It is suggested that the product of orf587 is an enzyme of C₁ metabolism and is unlikely to be a regulatory DNA-binding protein. Orf157 potentially encodes an unidentified basic protein, but the protein sequence is not conserved in other plants.     

Hypothetical gene C18orf42 encodes a novel protein kinase A‐binding protein

The substrate specificity of cAMP‐dependent protein kinase A (PKA) is controlled by its interaction with the A‐kinase anchoring protein (AKAP) family. Individual AKAP members are localized to particular intracellular sites and tether PKA specifically to the subcellular compartments where target substrates exist. Here, we report that the human hypothetical gene C18orf42 encodes a novel PKA‐binding protein that potentially regulates PKA–AKAP interactions. C18orf42 is expressed preferentially in neural tissues. Functional motif searching predicted that C18orf42 may encode a short protein that contains a putative PKA‐binding motif. To confirm this possibility, we applied the CRISPR/Cas9 genome‐editing system to incorporate the FLAG tag into the C‐terminus of the endogenous C18orf42 protein in the mouse neural cell line Neuro2a. Immunoprecipitation and immunoblotting using anti‐FLAG antibody showed translation of the endogenous C18orf42 protein and the physical interaction of the C18orf42 protein with PKA subunits. Immunoprecipitation and pull‐down assays showed that C18orf42 binds specifically to the type II regulatory subunits of PKA. Unlike the expression of many AKAPs, that of C18orf42 could block the AKAP‐mediated subcellular localization of PKA. These findings suggest that C18orf42 may be a novel PKA signaling gene that serves as an endogenous disruptor peptide for PKA–AKAP interactions.     

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster

 The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus. Received: 17 November 1995/16 January 1996     

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-β-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species. Received: 5 February / 20 March 2000     

Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system

The ability to perform multiple gene deletions is an important tool for conducting functional genomics. We report the development of a sequential gene deletion protocol for the filamentous fungus Aspergillus nidulans using the Cre/loxP recombinase system of bacteriophage P1. A recyclable genetic marker has been constructed by incorporating loxP direct repeats either side of the Neurospora crassa pyr-4 gene (encodes orotidine 5′-monophosphate decarboxylase) which is able to complement the A. nidulans pyrG89 mutation. This construct can be directed to delete specific genomic regions by attaching flanking sequences corresponding to the desired target. The pyr-4 marker can subsequently be eliminated by Cre-catalysed recombination between the loxP sites. The recombinase gene (cre), which has been placed under the control of the A. nidulans xlnA (xylanase A) gene promoter thus providing a means to switch on (xylose induction) or off (glucose repression) recombinase expression, has been integrated into the genome of an A. nidulans mutant strain defective in orotidine 5′-monophosphate decarboxylase activity (pyrG89). We demonstrate the effectiveness of our deletion system by sequentially deleting two genes, yellow (yA) and white (wA), involved in the synthesis of conidial pigment.     

Sequence analysis of RNA 2 and RNA 3 of lilac leaf chlorosis virus: a putative new member of the genus Ilarvirus

RNA 2 and RNA 3 of lilac leaf chlorosis virus (LLCV) were sequenced and shown to be 2,762 nucleotides (nt) and 2,117 nts in length, respectively. RNA 2 encodes a putative 807-amino-acid (aa) RNA-dependent RNA polymerase associated protein with an estimated M _r of 92.75 kDa. RNA 3 is bicistronic, with ORF1 encoding a putative movement protein (277 aa, M _r 31.45 kDa) and ORF2 encoding the putative coat protein (221 aa, M _r 24.37 kDa). The genome organization is similar to that typical for members of the genus Ilarvirus. Phylogenetic analyses indicate a close evolutionary relationship between LLCV, ApMV, and PNRSV.     

Allelism of SNQ1 and ATR1, genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide an aminotriazole

Summary SNQ1 gene function is required for the expression of resistance to 4NQO in wild-type yeast. The sequence of a 3.7 kb yeast DNA containing the gene SNQ1 was determined. The SNQ1 gene consists of an open reading frame of 1641 bp and encodes, according to the hydrophobicity analysis of the putative protein, a transmembrane protein of 547 amino acids. Homology searches in yeast genome databanks revealed a 100% sequence homology with gene ATR1 which controls resistance to aminotriazole in S. cerevisiae. Pre-treatment of wild-type yeast, but not of snq1-0::LEU2 disruption mutants, with sublethal doses of aminotriazole induced hyper-resistance to 4-nitroquinoline-N-oxide. Partial deletion of the nucleotide sequence coding for a putative ATP-binding site has no, or little, influence on resistance to 4NQO whereas total deletion of the region coding for this ATP-binding domain leads to 4NQO-sensitive nullmutants.Abbreviation 4NQO 4-nitroquinoline-N-oxide - aminotriazole 3-amino-1,2,4-triazole - HYR hyper-resistance