Deletion mapping of theniiAniaD gene region ofAspergillus nidulans

Tomsett, A. Brian; Cove, David J.

doi:10.1017/s001667230001925x

Cited by 64 publications

(31 citation statements)

References 23 publications

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“…The crnA mutants of E. nidulans were first isolated as a class of chlorate-resistant strains that were able to utilize nitrate as the sole nitrogen source (8). The crnA mutants were subsequently found to be defective in nitrate uptake at the conidiospore and young mycelial stages (7).…”

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confidence: 99%

A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

Zhou

Trueman²,

Boorer

et al. 2000

Journal of Biological Chemistry

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We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H ؉ -coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H ؉ , and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two MichaelisMenten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.

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confidence: 99%

A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

Zhou

Trueman²,

Boorer

et al. 2000

Journal of Biological Chemistry

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“…As A. nidulans recipient strain the nia" deletion mutant ANGW1103, which is an are derivative of strain SAA1017 (yA2, argB2, niaD26) (T. Goossen, personal communication), was used. The nia-phenotype of SAA1017 is due to a deletion in the niaD gene (Tomsett and Cove 1979). CaC12-PEG-mediated transformation was performed using 2x10 protoplasts and 5 lig of plasmid DNA.…”

Section: Methodsmentioning

confidence: 99%

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

et al. 1995

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The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a niadeletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia-mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli P-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.C. M. J. Pietersel J. van't Klooster G. C. M. van den Berg-Velthuis F. Govers (2i)

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“…nidulans strains and phenotypic studies of mutants. The strains, media, growth conditions and phenotypic tests used in this study were as described previously (Cove, 1966 ;Tomsett & Cove, 1979 ;Garde et al, 1995). The niaD mutations created by Garde et al (1995) are referred to here as follows : niaD3004 (niaDE850P), niaD3005 (niaDEA850\1PP), niaD-3010 (niaDH654A).…”

Section: Methodsmentioning

confidence: 99%

“…Protoplasts of A. nidulans strain niiA niaD∆503 (pabaA1) (Tomsett & Cove, 1979) were made by the method of Johnstone et al (1990), including modifications of Garde et al (1995). DNA (5 µg) was added to 100 µl protoplasts in a 0n4 mm electroporation cuvette and incubated on ice for 1 min.…”

Section: Methodsmentioning

confidence: 99%

“…A. nidulans offers such a system (Garde et al, 1995). In this fungus, the structural genes for NaR (niaD) and nitrite reductase (niiA) are tightly linked and are divergently transcribed (Tomsett & Cove, 1979 ;Johnstone et al, 1990) ; transformation of a deletion strain allows selection for niiA + arising from homologous targeted integration (Garde et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

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Structure–function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo

Hall

Tomsett

2000

Microbiology

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Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a twoelectron transfer. In fungi, the electron donor for NaR is NADPH whereas plants can have two enzymes, NADH :NaR and a bispecific NAD(P)H :NaR. PCR mutagenesis was employed to introduce mutations into the niaD gene of Aspergillus nidulans in order to identify residues involved in co-enzyme specificity. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specificity : the new enzyme had high levels of NADH :NaR activity in vitro, whilst all NADPH-associated activity was lost. However, strains carrying this mutation did not grow on nitrate. Enzyme assays suggested that this was not due to inhibition of the mutant enzyme by NADPH. All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity. Sequence analysis of these revertants showed that they all contained a single amino acid change at Asp-813, suggesting that this position is crucial to coenzyme specificity. Further studies have shown that the mutant enzyme was not protected from deactivation by either co-factor in cell-free extracts (unlike the wild-type), and that induction of the glucose-6-phosphate dehydrogenase occurred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions.

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Deletion mapping of theniiAniaD gene region ofAspergillus nidulans

Cited by 64 publications

References 23 publications

A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

Structure–function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo

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