Skip to main content
SpringerLink
Log in

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa

  • Original Articles
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akins RA, Lambowitz AM (1985) General method for cloning Neurospora crassa nuclear genes by complementation of mutants. Mol Cell Biol 5:2272–2278

    Google Scholar 

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

    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 

  • Burger G, Strauss J, Scazzocchio C, Lang F (1991) nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions. Mol Cell Biol 11:5746–5755

    Google Scholar 

  • Cambareri EB, Jensen BJ, Schabtach E, Selker EU (1989) Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571–1575

    Google Scholar 

  • Campbell WH (1986) Properties of bromphenol blue as an electron donor for higher plant NADH: nitrate reductase. Plant Physiol 82:729–732

    Google Scholar 

  • Cherniack AD, Garriga G, Kittle JD, Lambowitz AM (1990) Function of Neurospora mitochondrial tyrosyl tRNA-synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases. Cell 62:745–755

    Google Scholar 

  • Cove DJ, Pateman JA (1969) Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. J Bacteriol 97:1374–1378

    Google Scholar 

  • Davis RH, deSerres F (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol 17A:79–143

    Google Scholar 

  • Ebbole D, Sachs MS (1990) A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet Newslett 37:17–18

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Fu Y, 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, Marzluf GA (1990a) nit-2, the major nitrogen 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, 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 

  • Garrett RH, Cove DJ (1967) Formation of NADPH-nntrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants. Mol Gen Genet 149: 179–186

    Google Scholar 

  • Garrett RH, Nason A (1967) Involvement of a b-type cytochrome in the assimilatory nitrate reductase of Neurospora crassa. Proc Natl Acad Sci USA 58:1603–1610

    Google Scholar 

  • Hawker KL, Montague P, Marzluf GA, Kinghorn JR (1991) Heterologous expression and regulation of the Neurospora crassa nit-4 pathway-specific regulatory gene for nitrate assimilation in Aspergillus nidulans. Gene 100:237–240

    Google Scholar 

  • Hurlburt BK, Garrett RH (1988) Nitrate assimilation in Neurospora crassa: Enzymatic and immunoblot analysis of wild-type and nit mutant protein -products in nitrate-induced and glutamine-repressed cultures. Mol Gen Genet 211:35–40

    Google Scholar 

  • Jarai G, Marzluf GA (1991a) Sulfate transport in Neurospora crassa: regulation, turnover, and cellular localization of the CYS-14 protein. Biochemistry 30:4768–4773

    Google Scholar 

  • Jarai G, Marzluf GA (1991b) Generation of new mutants of nmr, the negative-acting nitrogen regulatory gene of Neurospora crassa, by repeat induced mutation. Curr Genet 20:283–288

    Google Scholar 

  • Kinghorn JRS, Campbell EI (1989) Amino acid sequence relationships between bacterial, fungal and plant nitrate reductase and nitrite reductase proteins. In: Wray JL, Kinghorn JRS (eds) Molecular and genetic aspects of nitrate assimilation. Oxford Science Publications, Oxford, UK, pp 385–403

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Le KHD, Lederer F (1983) On the presence of a heme-binding domain homologous to cytochrome b5 in Neurospora crassa assimilatory nitrate reductase. EMBO J 2:1909–1914

    Google Scholar 

  • Marathe S, Connerton IF, Fincham JRS (1990) Duplication-nduced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product. Mol Cell Biol 10:2638–2644

    Google Scholar 

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

    Google Scholar 

  • Metzenberg RL, Baisch TJ (1981) An easy method for preparing Neuospora DNA. Neurospora Newslett 28:20–21

    Google Scholar 

  • Okamoto PM, Fu YH, Marzluf GA (1991) Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet 227:213–223

    Google Scholar 

  • Perrine KG, Marzluf GA (1986) Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa. Curr Genet 10:677–684

    Google Scholar 

  • Premakumar R, Sorger GJ, Gooden D (1978) Stability of messenger RNA for nitrate reductase in Neurospora crassa. Biochim Biophys Acta 519:272–278

    Google Scholar 

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

    Google Scholar 

  • Riele HT, Maandag ER, Berns A (1992) Hingly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci USA 89:5128–5132

    Google Scholar 

  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn TG, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

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

    Google Scholar 

  • Selker EU (1990) Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet 24:579–613

    Google Scholar 

  • Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci USA 85:687–6874

    Google Scholar 

  • Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51:741–752

    Google Scholar 

  • Staben C, Jensen B, Singer M, Pollock J, Schechtman M, Kinsey J, Selker ELF (1989) Use of a bacterial hygromycin B resistance gene as a dominant marker in transformation. Fungal Genet Newslett 36:7981

    Google Scholar 

  • Subramanian KN, Sorger GJ (1972) Regulation of nitrate reductase in Neurospora crassa: regulation of transcription and translation. J Bacteriol 110:547–553

    Google Scholar 

  • Tomsett AB, Garrett RH (1980) The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa. Genetics 95:649–660

    Google Scholar 

  • Tomsett AB, Garrett RH (1981) Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase. Mol Gen Genet 184:183–190

    Google Scholar 

  • Yuan G, Fu Y, Marzluf GA (1991) nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol Cell Biol 11:5735–5745

    Google Scholar 

Download references

Author information

Author notes

  1. Patricia M. Okamoto

    Present address: Worcester Foundation for Experimental Biology, Shrewsbury, MA, USA

  2. George A. Marzluf

    Present address: Department of Biochemistry, Biological Sciences, Room 776, The Ohio State University, 484 West 12th Avenue, 43210, Colombus, OH, USA

Authors and Affiliations

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

    Patricia M. Okamoto & George A. Marzluf

  2. Department of Biology, University of Virginia, Charlottesville, VA, USA

    Reginald H. Garrett

Authors
  1. Patricia M. Okamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reginald H. Garrett
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. George A. Marzluf
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C.A.M.IJ. van den Hondel

Rights and permissions

Reprints and permissions

About this article

Cite this article

Okamoto, P.M., Garrett, R.H. & Marzluf, G.A. Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa . Molec. Gen. Genet. 238, 81–90 (1993). https://doi.org/10.1007/BF00279534

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00279534

Key words

Search

Navigation