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

Abstract

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.The assimilation of nitrate is a highly regulated process in ascomycetes (13,18,20). The product of gene nmr-J (Fungal Stock Collection no. 3931, Fungal Genetics Stock Center [FGSC], Department of Microbiology, University of Kansas Medical Center, Kansas City, Kans.; allelic with MS5 FGSC 4504) in the presence of glutamine is thought to repress transcription of the structural genes for nitrate reductase (nit-3) and nitrite reductase (nit-6) and of genes controlling a number of other nitrogen-scavenging metabolic pathways in Neurospora crassa (7,16,17). The product of gene nit-2 (FGSC 982, 983) of N. crassa turns on nitrogen-scavenging metabolic pathways in the presence of a pathway-specific inducer and a positive effector when glutamine is absent from the medium (13,20). In the case of the assimilation of nitrate in N. crassa, the specific inducer is nitrate or nitrite (13,20), and the metabolic pathway-specific effector is the product of gene nit4 (nit-5 and nit4 are probably allelic) (13).In Aspergillus nidulans there is a similar system, but, unlike the situation in N. crassa, no mutations in a gene equivalent to nmr-J have been found, suggesting to Marzluf and Fu (G. from ours but presumably contains the same gene. Preliminary reports of both Marzluf's and our work have appeared in the abstracts of the 16th International Genetics Congress in Toronto, Ontario, Canada, 20-27 August 1988.) Isolation of a cosmid that complements nonrepressible nmr-1 and MS5 mutant strains. Nitrate reductase reduces nontoxic chlorate to toxic chlorite; hence, nitrate reductasecontaining Neurospora cells commit suicide on chloratecontaining medium. The synthesis of nitrate reductase is repressed in the presence of adequate leVels of glutamine in wild-type Neurospora cells; hence, on glutamine-plus-chlorate-containing medium, wild-type Neurospora cells will survive. The wild-type allele of MS5 (nmr-J) is needed for the repression by glutamine of the synthesis of nitrate reductase; hence, strains mutated in this gene will commit suicide on medium containing glutamine plus chlorate, unlike their wild-type or complemented counterparts. The Neurospora genomic library of Vollmer and Yanofsky (19) was made in cosmid pSV50, which contains a benomyl resistance gene. This library was screened by making DNA minipreparations (4, 10) of each entry, pooling equal porti...

Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa

“…Another gene, nmr (for nitrogen metabolic regulation), appears to be involved in nitrogen control; nmr mutants are insensitive to repression by NH + or glutamine and constitutively express the nitrogen-related structural genes. NMR appears to be a negative-acting nitrogen regulatory protein that acts in nitrogen catabolite repression (Fu, Young & Marzluf, 1988;Dunn-Coleman, Tomsett & Garret, 1981;Sorger et al, 1989;Xiao, Fu & Marzluf, 1995).…”

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.…”

Molecular comparison of the negative-acting nitrogen control gene,nmr, inNeurospora crassa and otherNeurospora and fungal species

“…The nmr gene has been cloned (Fu et al, 1988;Sorger et al, 1989) and its complete nucleotide sequence determined (Young et al, 1990). The nmr gene itself is constitutively expressed at a low level and appears to encode a protein which contains 488 amino acids (Young et aL, 1990).…”