Molecular Cloning and Analysis of the Regulation of Nit-3, the Structural Gene for Nitrate Reductase in Neurospora crassa

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 23; pp. 8243 - 8247
• Main Authors: Fu, Ying-Hui, Marzluf, George A.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1987; National Acad Sciences
• Subjects: Biological and medical sciences; Biotechnology; Cloning, Molecular; Cosmids; DNA; Enzymes; Fundamental and applied biological sciences. Psychology; Gels; gene expression; Gene Expression Regulation; Genes; Genes, Fungal; Genetic engineering; Genetic technics; genetics; Methods. Procedures. Technologies; Molecular cloning; Neurospora; Neurospora - genetics; Neurospora crassa; Neurospora crassa - genetics; Nitrate Reductase; Nitrate Reductases; Nitrate Reductases - genetics; Nitrates; Nitrogen; Nitrogen Fixation; Nitrogen Fixation - genetics; nitrogen metabolism; Polymorphism, Restriction Fragment Length; Repression; RNA; RNA, Messenger; RNA, Messenger - genetics; Transcription, Genetic; Fungi; Regulation(control); Gene; Nitrate reductase; Ascomycetes; Neurospora crassa; Nitrogen; Molecular cloning; Gene expression; Metabolism; Thallophyta
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.23.8243

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 ≈ 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 ≈ 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.