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Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

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Summary

The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.

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References

  • Aviv H, Leder P (1972) Purification of biologically active globin messager RNA by chromatography on oligothymidylic acidcellulose. Proc Natl Acad Sci USA 69:1408–1412

    Google Scholar 

  • Bahns M, Garrett R (1980) Demonstration of de novo synthesis of Neurospora crassa nitrate reductase during induction. J Biol Chem 255:690–693

    Google Scholar 

  • Berk AJ, Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721–732

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    CAS  PubMed  Google Scholar 

  • Bossinger J, Cooper TG (1977) Molecular events associated with induction of arginase in Saccharomyces cerevisae. J Bacteriol 131:163–173

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Calza R, Huttner E, Vincentz M, Rouze P, Galangau F, Vancheret H, Cherel I, Merger C, Kronenberger J, Caboche M (1987) Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants. Mol Gen Genet 209:552–562

    Google Scholar 

  • Campbell WH, Kinghorn JR (1990) Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem Sci 15:315–319

    Google Scholar 

  • Cheng CH, Dewdney J, Kleinhofs A, Goodman HM (1986) Cloning and nitrate induction of nitrate reductase mRNA. Proc Natl Acad Sci USA 83:6825–6828

    Google Scholar 

  • Crawford NM, Smith M, Bellissimo D, Davis RW (1988) Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci USA 85:5006–5010

    Google Scholar 

  • Cybis J, Weglenski P (1972) Arginase induction in Aspergillus nidulans. The appearance and decay of the coding capacity of messenger. Eur J Biochem 30:262–268

    Google Scholar 

  • Daniel-Vedele F, Dorbe M-F, Caboche M, Roze P (1989) Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. Gene 85:371–380

    Google Scholar 

  • Fernandez E, Schnell R, Ranum LPW, Hussey SC, Seflow CD, Lefebvre PA (1989) Isolaton and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 86:6449–6453

    Google Scholar 

  • Fu Y-H, Marzluf GA (1987a) Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase Neurospora crassa. Proc Natl Acad Sci USA 84:8243–8247

    Google Scholar 

  • Fu Y-H, Marzluf GA (1987b) Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa. Mol Cell Biol 7:1691–1696

    Google Scholar 

  • Fu Y-H, Marzluf GA (1988) Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa. J Bacteriol 170:657–661

    Google Scholar 

  • Fu Y-H, Marzluf GA (1990a) nit-2, the major regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol 10:1056–1065

    Google Scholar 

  • Fu Y-H, Marzluf GA (1990b) nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc Natl Acad Sci USA 87:5331–5335

    Google Scholar 

  • Fu Y-H, Young JL, Marzluf GA (1988) Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa. Mol Gen Genet 214:74–79

    Google Scholar 

  • Fu Y-H, Kneessi JY, Marzluf GA (1989) Isolation of nit-4, the minor regulatory gene which mediates nitrate induction in Neurospora crassa. J Bacteriol 171:4067–4070

    Google Scholar 

  • Galangau F, Cherel I, Deng M, Meyer C, Moureaux T, Rouze P, Vaucheret H, Vedele F, Vincentz M, Caboche M (1989) Nitrate reductase expression in tobacco and tomato. Curr Top Plant Biochem Physiol 7:26–34

    Google Scholar 

  • Garrett RH, Cove DJ (1976) Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants. Mol Gen Genet 149:179–186

    Google Scholar 

  • Goldberg AL, Dice JF (1974) Intracellular protein degradation in mammalian and bacterial cells. Annu Rev Biochem 43:835–869

    Google Scholar 

  • Gowri G, Campbell WH (1989) cDNA clones for corn leaf NADH nitrate reductase and chloroplast NAD(P)H:glyceraldehyde-3phosphate dehydrogenase. Plant Physiol 90:792–798

    Google Scholar 

  • Guiard B, Lederer F (1979) The “cytochrome b5 fold”: structure of a novel protein superfamily. J Mol Biol 135:639–650

    Google Scholar 

  • Hackett CS, Novoa WB, Orzols J, Strittmatter P (1986) Identification of the essential cysteine residue of NADH-cytochrome b5 reductase. J Biol Chem 261:9854–9857

    Google Scholar 

  • Hackett CS, Novoa WB, Read Kensil C, Strittmatter P (1988) NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110. J Biol Chem 263:7539–7543

    Google Scholar 

  • Hanson MA, Marzluf MA (1975) Control of the synthesis of a single enzyme by multiple regulatory circuits in Neurospora crassa. Proc Natl Acad Sci USA 72:1240–1244

    Google Scholar 

  • Hyde GE, Wilberding JA, Meyer AL, Campbell ER, Campbell WH (1989) Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH: nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase. Plant Mol Biol 13:233–246

    Google Scholar 

  • Johnstone IL, McCabe PC, Greaves P, Cole GE, Brow MAD, Gurr SJ, Unkles SE, Clutterbuck AJ, Kinghorn JR, Innis M (1990) The isolation and characterization of the crnA—niiA—niaD gene cluster for nitrate assimilation in the filamentous fungus Aspergillus nidulans. Gene 90:181–192

    Google Scholar 

  • Ketter JS, Jarai G, Fu Y-H, Marzluf GA (1991) Nucleotide sequence, messenger RNA stability, and DNA recognition elements of cys-14, the structural gene for sulfate permease II in Neurospora crassa. Biochemistry 30:1780–1787

    Google Scholar 

  • Kim HDL, Lederer F (1983) On the presence of a heme-binding domain homologous to cytochrome b5 in Neurospora crassa assimilatory nitrate reductase. EMBO J 2:1909–191

    Google Scholar 

  • Lee H, Fu Y-H, Marzluf GA (1990) Nucleotide sequence and DNA recognition elements of ale, the structural gene which encodes allantoicase, a purine cataobolic enzyme of Neurospora crassa. Biochemistry 29:8779–8787

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Marzluf GA (1981) Regulation of nitrogen metabolism and gene expression in fungi. Microbiol Rev 45:437–461

    Google Scholar 

  • Marzluf GA, Fu Y-H (1988) Genetic and metabolic regulation of nitrogen metabolism in Neurospora crassa. In: Sanchez-Esquivel S (ed) Nitrogen source control of microbial processes, CRC Press, Boca Raton, Fla

    Google Scholar 

  • Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461

    Google Scholar 

  • Premakumar R, Sorger GJ (1979) Nitrogen metabolite repression of nitrate reductase in Neurospora crassa. J Bacteriol 137:1119–1126

    Google Scholar 

  • Premakumar R, Sorger GJ, Gooden D (1978) Stability of messenger RNA for nitrate reductase in Neurospora crassa. Biochim Biophys Acta 519:275–278

    Google Scholar 

  • Reinert WR, Patel VB, Giles NH (1981) Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency in qa-1 function. Mol Cell Biol 1:829–835

    Google Scholar 

  • Roberts AN, Berlin V, Hager KM, Yanofsky C (1988) Molecular analysis of a Neurospora crassa gene expressed during conidiation. Mol Cell Biol 8:2411–2418

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    CAS  PubMed  Google Scholar 

  • Solomonson LP, Barber MJ (1990) Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41:225–253

    Google Scholar 

  • Sorger GJ, Debanne MT, Davies J (1974) Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora crassa. Biochem J 140:395–403

    Google Scholar 

  • Yoo M, Steggles AW (1988) The complete nucleotide sequence of human liver cytochrome b5 mRNA. Biochem Biophys Res Commun 156:576–580

    Google Scholar 

  • Young JL, Jarai G, Fu Y-H, Marzluf GA (1990) Nucleotide sequence and analysis of nmr, a negative-acting regulatory gene in the nitrogen circuit of Neurospora crassa. Mol Gen Genet 222:120–128

    Google Scholar 

  • Yubisui T, Miyata T, Iwanaga S, Tamura M, Takeshita M (1986) Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes. J Biochem 94:407–422

    Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry, The Ohio State University, 43210, Columbus, OH, USA

    Patricia M. Okamoto, Ying-Hui Fu & George A. Marzluf

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  1. Patricia M. Okamoto
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  2. Ying-Hui Fu
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  3. George A. Marzluf
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Communicated by C.A. van den Handel

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Okamoto, P.M., Fu, YH. & Marzluf, G.A. Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Molec. Gen. Genet. 227, 213–223 (1991). https://doi.org/10.1007/BF00259673

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  • DOI: https://doi.org/10.1007/BF00259673

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