Analysis of conventional and in vitro generated mutants of nmr, the negatively acting nitrogen regulatory gene of Neurospora crassa

Abstract

The nmr gene is the major negative regulatory gene in the nitrogen control circuit of Neurospora crassa, which, together with positive regulatory genes, governs the expression of multiple unlinked structural genes of the circuit. Possible functional domains of the NMR protein were investigated by mutational analyses using three different approaches. First, the polymerase chain reaction was used to clone the nmr locus from two conventional mutants, V2M304 and MS5, and the mutant amino acid codons were identified. A single point mutation was shown to be responsible for the mutant phenotype in each of these strains. The V2M304 allele contains a nonsense codon, and in the MS5 allele an aspartate has been substituted for glycine at residue 386. Our second approach studied possible functionally important regions in the nmr gene by the use of site-directed mutagenesis. The region containing the naturally occurring substitution in MS5 appears to be essential for function whereas a region in the N-terminal part of the protein does not seem important for NMR function. Finally, over 50% of the protein coding region was randomly mutagenized and amino acid residues that are essential for function and others that are functionally unimportant were identified.