Nitrate Reductase Gene Expression Research Articles

Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa.

The nit-3 gene of Neurospora crassa encodes the enzyme nitrate reductase and is regulated by nitrogen catabolite repression and by specific induction with nitrate. The nit-3 gene was isolated from a cosmid-based genomic library by dual selection for benomyl resistance and for the ability to complement a nit-3 mutant strain using the sibling-selection procedure. The nit-3 gene was subcloned as a 3.8-kilobase DNA fragment from a cosmid that carried an approximately 40-kilobase N. crassa DNA insert. A restriction fragment length polymorphism analysis revealed that the cloned segment displayed tight linkage to two linkage-group-4 markers that flank the genomic location of nit-3. RNA gel blot analyses of RNA from wild-type and various mutant strains were carried out to examine the molecular mechanism for regulation of nit-3 gene expression. The nit-3 gene was transcribed to give a large mRNA of approximately 3.4 kilobases, the expected size to encode nitrate reductase. The nit-3 gene was only expressed in wild-type cells subject to simultaneous nitrogen derepression and nitrate induction. A mutant of nit-2, the major nitrogen regulatory gene of Neurospora, did not have detectable levels of nit-3 gene transcripts under the exact conditions in which these transcripts were highly expressed in wild type. Similarly, a mutant of nit-4, which defines a minor positive-acting nitrogen control gene, failed to express detectable levels of the nit-3 transcript. Nitrate reductase gene expression was partially resistant to nitrogen repression in a mutant of the nmr gene, which appears to be a regulatory gene with a direct role in nitrogen catabolite repression. Results are presented that suggest that the enzyme glutamine synthetase does not serve any regulatory role in controlling nitrate reductase gene expression.

Immunoelectrophoretic determination of nitrate reductase in Neurospora crassa

Highly selective and sensitive methods for determining nitrate reductase levels independent of any expression of its enzymatic activity have been developed, based on immunoelectrophoretic procedures. Specific antisera raised against Neurospora crassa NADPH-nitrate reductase inhibited the overall NADPH-nitrate reductase activity of this enzyme as well as partial activities of the electron transfer process associated with the nitrate reductase reaction. Three immunological techniques were employed to detect the presence of nitrate reductase-related protein in various Neurospora extracts: (i) a protection against inhibition assay, (ii) rocket immunoelectrophoresis, and (iii) crossed immunoelectrophoresis. These three methods were then applied to an analysis of nitrate reductase gene expression in the nit mutants of N. crassa.