Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa

Abstract

Expression of the structural genes of the nitrogen control circuit of Neurospora crassa is regulated by the positive-acting nit-2 control gene and by the negative-acting nmr control gene. Nitrate reductase is expressed in a constitutive fashion in nmr mutant strains, which appear to be largely insensitive to nitrogen catabolite repression. Thus, nmr mutants are sensitive to chlorate in the presence of ammonia or glutamine, whereas the wild type is chlorate resistant under these conditions. A cosmid library was screened for the presence of the nmr+ gene by the sib selection procedure, and a single cosmid was isolated which transforms the nmr mutant to chlorate resistance at a high frequency. A restriction fragment length polymorphism analysis revealed that the cloned DNA segment maps to the precise genomic location of nmr. Northern blot analyses revealed that the nmr gene is itself not regulated but is expressed constitutively to give a single transcript of approximately 1.8 kb.