Molecular Cloning, Characterization, and Nucleotide Sequence of Nit-6, the Structural Gene for Nitrite Reductase in Neurospora Crassa

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1993 Apr 1

PMID 8096840

Citations 20

Authors

G E Exley

J D Colandene

R H Garrett

Affiliations

Soon will be listed here.

Abstract

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.

Citing Articles

Optogenetic control of horizontally acquired genes prevent stuck fermentations in yeast.

Figueroa D, Ruiz D, Tellini N, De Chiara M, Kessi-Perez E, Martinez C Microbiol Spectr. 2025; 13(2):e0179424.

PMID: 39772912 PMC: 11792454. DOI: 10.1128/spectrum.01794-24.

Nitrogen Journey in Plants: From Uptake to Metabolism, Stress Response, and Microbe Interaction.

Zayed O, Hewedy O, Abdelmoteleb A, Ali M, Youssef M, Roumia A Biomolecules. 2023; 13(10).

PMID: 37892125 PMC: 10605003. DOI: 10.3390/biom13101443.

Thermophilic methane oxidation is widespread in Aotearoa-New Zealand geothermal fields.

Houghton K, Carere C, Stott M, McDonald I Front Microbiol. 2023; 14:1253773.

PMID: 37720161 PMC: 10502179. DOI: 10.3389/fmicb.2023.1253773.

The First Step of Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions.

Wajmann S, Hercher T, Buchmeier S, Hansch R, Mendel R, Kruse T Microorganisms. 2020; 8(4).

PMID: 32272807 PMC: 7232280. DOI: 10.3390/microorganisms8040534.

Light-regulated promoters for tunable, temporal, and affordable control of fungal gene expression.

Fuller K, Dunlap J, Loros J Appl Microbiol Biotechnol. 2018; 102(9):3849-3863.

PMID: 29569180 PMC: 5897137. DOI: 10.1007/s00253-018-8887-7.

References

Amy N, Garrett R . Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa. J Bacteriol. 1980; 144(1):232-7. PMC: 294629. DOI: 10.1128/jb.144.1.232-237.1980. View

Fu Y, Kneesi J, Marzluf G . Isolation of nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in Neurospora crassa. J Bacteriol. 1989; 171(7):4067-70. PMC: 210163. DOI: 10.1128/jb.171.7.4067-4070.1989. View

Fu Y, Young J, Marzluf G . Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa. Mol Gen Genet. 1988; 214(1):74-9. DOI: 10.1007/BF00340182. View

ROBERTS A, BERLIN V, Hager K, Yanofsky C . Molecular analysis of a Neurospora crassa gene expressed during conidiation. Mol Cell Biol. 1988; 8(6):2411-8. PMC: 363439. DOI: 10.1128/mcb.8.6.2411-2418.1988. View

Jarai G, Marzluf G . Analysis of conventional and in vitro generated mutants of nmr, the negatively acting nitrogen regulatory gene of Neurospora crassa. Mol Gen Genet. 1990; 222(2-3):233-40. DOI: 10.1007/BF00633823. View

Vollmer S, Yanofsky C . Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986; 83(13):4869-73. PMC: 323844. DOI: 10.1073/pnas.83.13.4869. View

Tomsett A, Garrett R . The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa. Genetics. 1980; 95(3):649-60. PMC: 1214252. DOI: 10.1093/genetics/95.3.649. View

Fu Y, Marzluf G . Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa. Proc Natl Acad Sci U S A. 1987; 84(23):8243-7. PMC: 299518. DOI: 10.1073/pnas.84.23.8243. View

Dunn-Coleman N, Garrett R . The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa. Mol Gen Genet. 1980; 179(1):25-32. DOI: 10.1007/BF00268442. View

10.

Fu Y, Marzluf G . Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa. Mol Cell Biol. 1987; 7(5):1691-6. PMC: 365269. DOI: 10.1128/mcb.7.5.1691-1696.1987. View

11.

Tomsett A, Garrett R . Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase. Mol Gen Genet. 1981; 184(2):183-90. DOI: 10.1007/BF00272903. View

12.

Campbell W, Kinghorn K . Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem Sci. 1990; 15(8):315-9. DOI: 10.1016/0968-0004(90)90021-3. View

13.

Pearson W . Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 1990; 183:63-98. DOI: 10.1016/0076-6879(90)83007-v. View

14.

Tomsett A, Dunn-Coleman N, Garrett R . The regulation of nitrate assimilation in Neurospora crassa: the isolation and genetic analysis of nmr-1 mutants. Mol Gen Genet. 1981; 182(2):229-33. DOI: 10.1007/BF00269662. View

15.

Werner M, Feller A, Messenguy F, PIERARD A . The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell. 1987; 49(6):805-13. DOI: 10.1016/0092-8674(87)90618-0. View

16.

Lafferty M, Garrett R . Purification and properties of the Neurospora crassa assimilatory nitrite reductase. J Biol Chem. 1974; 249(23):7555-67. View

17.

Prodouz K, Garrett R . Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. J Biol Chem. 1981; 256(18):9711-7. View

18.

Peakman T, Crouzet J, Mayaux J, Busby S, Mohan S, Harborne N . Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome. Eur J Biochem. 1990; 191(2):315-23. DOI: 10.1111/j.1432-1033.1990.tb19125.x. View

19.

Garrett R, Amy N . Nitrate assimilation in fungi. Adv Microb Physiol. 1978; 18:1-65. DOI: 10.1016/s0065-2911(08)60414-2. View

20.

Bahns M, Garrett R . Demonstration of de novo synthesis of Neurospora crassa nitrate reductase during induction. J Biol Chem. 1980; 255(2):690-3. View