Identification of the product of the major regulatory gene of the nitrogen control circuit of Neurospora crassa as a nuclear DNA-binding protein.

The nitrogen regulatory circuit of Neurospora crassa contains an entire set of unlinked structural genes which specify nitrogen catabolic enzymes. These genes are controlled as a group by a major regulatory gene, designated nit-2, which "turns on" their expression in a positive fashion. Moreover, expression of these same structural genes is repressed when the cells contain sufficient nitrogen; the active repressor metabolite has been identified as glutamine. It has been suggested that the nit-2 gene product is a protein which binds at a recognition sequence near each nitrogen-related structural gene to activate their expression. We have directly examined the nuclear proteins of Neurospora in order to attempt to detect th postulated nit-2 regulatory protein. Nonhistone nuclear proteins were isolated, labeled in vitro, and applied to DNA-cellulose and subsequently specifically eluted with glutamine. A single protein, whose molecular weight is approximately 22,000, was eluted from DNA-cellulose by glutamine but not by asparagine. This same protein was greatly reduced in quantity in the nonhistone proteins isolated from two nit-2 mutants; a third nit-2 mutant has a normal amount of this nuclear protein but it displays a slightly higher electrophoretic mobility than found in wild type. Finally, a revertant of a nit-2 mutant possesses approximatley 20 times as much of this protein as does the parental nit-2 mutant, an amount essentially equivalent to that found in wild type. The results suggest that the nit-2 control gene encodes this DNA-binding protein, which is postulated to play a major role in regulating expression of the structural genes of the nitrogen control circuit of Neurospora.