The regulation of nitrate assimilation in Neurospora crassa: The isolation and genetic analysis of nmr-1 mutants

Abstract

Four mutants of Neurospora crassa have been isolated which have altered regulation of nitrate reductase. They each carry a mutation which results in derepressed synthesis of nitrate reductase even in the presence of glutamine. They map to a single locus which has been designated nmr-1 and which is located between am and gln on linkage group VR. The mutations appear to affect only nitrate assimilation. The nit-2, nit-3 and nit-4/5 mutations are epistatic to nmr-1 since the double mutants have the single nit mutant phenotype. For nitrate reductase synthesis, the nmr-1 mutation is epistatic to am such that the double mutant is derepressed even in the presence of glutamate or glutamine. In all other respects however, the double mutant exhibits the am phenotype. We suggest therefore that the nmr-1 mutations do not directly affect the regulation of nitrate reductase at the level of transcription but instead act post-transcriptionally.

nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.

“…The N. crassa wild-type strain 740R231A and nit-2 mutant strains were obtained from the Fungal Genetics Stock Center (University of Kansas Medical Center). Cultures were grown in Vogels liquid medium supplemented as indicated for each experiment with shaking at 30'C as described (10)(11)(12)(13)(14).…”

“…It is important to note that these recognition sequences have only been identified in vitro and studies are needed to determine whether any or all of these sites function in vivo in controlling gene expression. The length of the individual footprints shows some variation; furthermore, three of the NIT2 binding sites appear to be bipartite-i.e., the footprints show two regions with protected and enhanced cleavages, separated by [10][11][12][13][14][15][16][17][18][19][20] bp that show the usual DNase I sensitivity. These observations suggest the possibility that two or more molecules of the NIT2 protein can bind at some of these elements, perhaps even in a cooperative manner.…”

“…The product of nit-2, the major positive-acting regulatory gene of the nitrogen circuit, is necessary for the expression of the structural genes of the circuit during conditions of nitrogen limitation (6)(7)(8)(9). A distinct unlinked regulatory gene, nmr (for "nitrogen metabolic regulation") acts in a negative manner (10)(11)(12)(13). The nit-2 gene and the nmr gene of N. crassa both appear to be directly involved in nitrogen catabolite repression, but the mechanism of their interaction is not yet understood.…”

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

“…Mutations in another gene, nmr (for 'nitrogen metabolite repression'), result in the loss of nitrogen repression; nitrate reductase and other related enzymes are expressed in nmr mutants when the cells have sufficient glutamine or ammonia to inhibit their synthesis in the wild type completely (Premakumar et al, 1980;Tomsett et al, 1981). These observations imply that the nmr gene product functions in some aspect of repression.…”

Relationship between two major immunoreactive forms of arginase in Neurospora crassa

“…Arginase levels are unaffected by the nit-2 mutation (15), indicating that the nit-2 regulatory mechanism is not responsible for nitrogen control of arginase levels. The nmr locus, which is responsible for nitrogen catabolite repression of nitrate assimilatory genes, also does not appear to regulate arginase levels in N. crassa (15,36).…”