Isolation of a gene that down-regulates nitrate assimilation and influences another regulatory gene in the same system.

Glutamine is the preferred source of nitrogen of Neurospora crassa. In its presence and that of the gene product of MS5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. In the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. We report here the isolation, from a cosmid bank, of a gene that complements MS5 and can also complement nit-2. We speculate that this result suggests an interaction between the MS5 and nit-2 gene products and that this is important in the regulation of nitrate assimilation.     

Identification and Characterization of a Nitrate Transporter Gene in Neurospora crassa

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.     

Molecular characterization of mutations of nlt-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.     

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa.

In the filamentous fungus Neurospora crassa, both the global-acting regulatory protein NIT2 and the pathway-specific regulatory protein NIT4 are required to turn on the expression of the nit-3 gene, which encodes nitrate reductase, the first enzyme in the nitrate assimilatory pathway. Three NIT2 binding sites and two NIT4 binding sites have been identified in the 1.3-kb nit-3 promoter region via mobility shift and footprinting experiments with NIT2-beta-galactosidase and NIT4-beta-Gactosidase fusion proteins. Quantitative mobility shift assays were used to examine the affinity of individual NIT2 binding sites for the native NIT2 protein present in N. crassa nuclear extracts. In vivo analysis of nit-3 promoter 5' deletion constructs and individual NIT2 and NIT4 binding-site deletions or mutations revealed that all of the NIT2 and NIT4 binding sites are required for the full level of expression of the nit-3 gene. A cluster of two NIT2 and two NIT4 binding sites located more than 1 kb upstream of the translational start site is required for nit-3 expression, and one NIT2 binding site and one NIT4 site, which are immediately adjacent to each other, are of particular functional importance. A significant NIT2-NIT4 protein-protein interaction might occur upon their binding to nearby sites.     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

Hypersensitive sites in the 5' promoter region of nit-3, a highly regulated structural gene of Neurospora crassa.

The nit-3 gene of the filamentous fungus Neurospora crassa encodes nitrate reductase, the enzyme which catalyzes the first step in nitrate assimilation. The nit-3 gene is subject to a high degree of regulation by metabolic inducers and repressors, and its expression requires two distinct trans-acting regulatory proteins. Hypersensitive sites in the 5' DNA sequence upstream of the nit-3 gene were mapped with the use of three different nucleases as molecular probes. Six hypersensitive sites, three of which are very strong, were detected at essentially identical positions by all three nucleases. The hypersensitive sites appear to develop in a constitutive fashion and are present under conditions in which the nit-3 structural gene is expressed but also when this gene is inactive, although these sites are considerably less prominent in cells subjected to nitrogen catabolite repression. The presence of the hypersensitive sites appears to depend upon both the positively acting NIT2 and the positively acting NIT4 regulatory proteins, which might play a role in positioning of chromatin protein.     

The Neurospora crassa genome: cosmid libraries sorted by chromosome

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.     

A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Physiological characterization of a Neurospora crassa mutant with impaired regulation of nitrate reductase.

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation.     

In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants.

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.