Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa

The nit-2 gene is the major nitrogen-regulatory gene of Neurospora crassa, and under conditions of nitrogen limitations, it turns on the expression of various unlinked structural genes which specify nitrogen-catabolic enzymes. The nit-2 gene was subcloned as a 6-kilobase (kb) DNA fragment from a cosmid that carried approximately a 40-kb N. crassa DNA insert. The nit-2 gene was localized in a DNA segment of approximately 3.5 kb and was shown to correspond to a unique DNA sequence located on linkage group 1. Several N. crassa nit-2 transformants were characterized and were found to possess significantly different levels of the regulated enzyme nitrate reductase. Northern blot analysis of RNA from various strains was carried out to determine whether the nit-2 gene was expressed constitutively or was itself subject to regulation. The results revealed that the nit-2 gene is transcribed to give a single large mRNA of approximately 3.5 kb. Expression of the nit-2 gene is regulated such that its transcript is present at a substantially higher level in cells which are limited for nitrogen than in cells growing under nitrogen-repressed conditions. However, the nit-2 gene is not controlled by autogenous regulation. The nit-2 gene was transcribed only at a low level in nmr-1 and in gln-1b, under both nitrogen-repressed and derepressed conditions, suggesting that these unlinked loci may exert a positive regulatory effect on nit-2.