In fungi, such as Aspergillus nidulans, the nitrate transport system (a permease) is encoded by a gene (crnA) distinct from the genes encoding nitrate reductase (niaD), and nitrite reductase (niiA), although all three of these genes are tightly clustered (Kinghorn, 1989). The crnA gene specifies a predicted polypeptide of 483 amino acids; a polypeptide that has 10 membrane-spanning helices (Unkles et al, 1991). The nitrate-assimilation gene cluster of Aspergillus fumigatus is very similar to that of Aspergillus nidulans. The physically linked niaD, niiA and crnA genes are induced by nitrate and repressed by ammonium at the transcriptional level in A. fumigatus (Amaar and Moore, 1998). Similarly the YNT1 gene encoding the nitrate transporter is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, respectively, in the yeast Hansenula polymorpha (Perez et al, 1997). YNT1 is expressed when the yeast is grown on nitrate and nitrite but not on ammonium (Perez et al, 1997).

The areA control gene is required for nitrogen metabolite (or ammonium) repression of the nitrate assimilation systems; the nirA gene is a control gene required for nitrate induction of nitrate assimilation systems (Kinghorn, 1989; Kudla et al, 1990). The Aspergillus nidulans AREA protein shares homology with the GLN3 protein of Saccharomyces cerevisiae, the Neurospora crassa NIT2 protein (Minehart and Magasanik, 1991), and the NRE protein of Penicillium chrysogenum (Haas et al, 1995); these proteins contain a single Cys2/Cys2-type zinc finger (Minehart and Magasanik, 1991; Haas et al, 1995). Consist with this, the areA- regulatory gene mutations of Aspergillus nidulans can in part be complemented by the heterologous regulatory gene nit-2 of Neurospora crassa (Davis and Hynes, 1987). Likewise the nre gene of Penicillium chrysogenum has been shown to be functional in N. crassa by heterologous complementation of a nit-2 mutant (Haas et al, 1995). The AREA protein binds specifically to the niaD promoter in Aspergillus (Lamb et al, 1997).

The niaD and niiA genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction (Punt at al, 1995). The A. nidulans NirA gene product may be functionally similar to the N. crassa NIT4 pathway specific regulatory protein (Chang et al, 1996).

The GLN3 gene of the yeast Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen (Minehart and Magasanik, 1991). Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase (Minehart and Magasanik, 1991). Note that Saccharomyces cerevisiae lacks genes encoding NR and NiR. Saccharomyces cerevisiae also lacks molybdopterin synthase homologues (Unkles et al, 1999). Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzyme (Unkles et al, 1999). Consequently, when Nicotiana tabacum nitrate reductase is expressed in Saccharomyces cerevisiae the haem and FAD domains are functional, but the molybdenum cofactor (MoCo) domain is inactive due to the absence of MoCo in yeast (Truong et al, 1991). Because the native form of the plant NR is dimeric when expressed in yeast, this suggest that MoCo is not involved in NR dimerization in higher plants (Truong et al, 1991).

In Aspergillus nidulans molybdopterin synthase is encoded by two genes: cnxG (encoding the small subunit) and cnxH (encoding the large subunit) (Unkles et al, 1999). In addition, the complex locus, cnxABC, is required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway (Unkles et al, 1997).

In fungi (including Aspergillus) the electron donor for NR is NADPH [EC 1.6.6.3], whereas plants can have two enzymes, NADH:NR and a bispecific NAD(P)H:NR (Hall and Tomsett, 2000). Mutations of the niaD gene have been found with altered co-enzyme specificity: high levels of NADH:NR activity with loss of NADPH-associated activity in vitro (Hall and Tomsett, 2000). However, Aspergillus nidulans strains carrying this mutation do not grow on nitrate (Hall and Tomsett, 2000).

Amaar YG, Moore MM 1998 Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. Curr. Genet. 33: 206-215.

Chang PK, Ehrlich KC, Linz JE, Bhatnagar D, Cleveland TE, Bennett JW 1996 Characterization of the Aspergillus parasiticus niaD and niiA gene cluster. Curr. Genet. 30: 68-75.

Davis MA, Hynes MJ 1987 Complementation of areA- regulatory gene mutations of Aspergillus nidulans by the heterologous regulatory gene nit-2 of Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 84: 3753-3757.

Haas H, Bauer B, Redl B, Stoffler G, Marzluf GA 1995 Molecular cloning and analysis of nre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr. Genet. 27: 150-158.

Hall N, Tomsett AB 2000 Structure-function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo. Microbiology 146: 1399-1406.

Kinghorn JR 1989 Genetic, biochemical, and structural organization of the Aspergillus nidulans crnA-niiA-niaD gene cluster. In (JL Wray, JR Kinghorn eds) "Molecular and Genetic Aspects of Nitrate Assimilation", Oxford Science Publications, Oxford, pp. 69-87.

Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF, Sibley S, Davies RW, Arst HN Jr 1990 The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J. 9: 1355-1364.

Lamb HK, Dodds AL, Swatman DR, Cairns E, Hawkins AR 1997 Deletion of the N-terminal region of the AREA protein is correlated with a derepressed phenotype with respect to nitrogen metabolite repression. J. Bacteriol. 179: 6649-6656.

Minehart PL, Magasanik B 1991 Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain. Mol. Cell. Biol. 11: 6216-6228.

Perez MD, Gonzalez C, Avila J, Brito N, Siverio JM 1997 The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate. Biochem. J. 321: 397-403.

Truong HN, Meyer C, Daniel-Vedele F 1991 Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae. Biochem. J. 278: 393-397.

Unkles SE, Hawker KL, Grieve C, Campbell EI, Montague P, Kinghorn JR 1991 crnA encodes a nitrate transporter in Aspergillus nidulans. Proc. Natl. Acad. Sci. U.S.A. 88: 204-208.

Unkles SE, Heck IS, Appleyard MV, Kinghorn JR 1999 Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants. J. Biol. Chem. 274: 19286-19293.

Unkles SE, Smith J, Kanan GJ, Millar LJ, Heck IS, Boxer DH, Kinghorn JR 1997 The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis. J. Biol. Chem. 272: 28381-28390.