1887

Abstract

The gene, encoding nitrate reductase, has been isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic transformations, giving frequencies of up to 200 transformants per μg DNA. Of 15 transformants analysed by Southern blots, six showed homologous integration whilst the remaining nine integrated at heterologous sites, indicating that the vector may be used reliably for both types of integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of defective mutants, and also for gene disruption experiments. Electrophoretic karyotype determination suggested at least 11 chromosomes ranging from 2 to 6 Mb, the total genome size being at least 37 Mb. The gene was assigned to chromosome V by Southern blot analysis. The gene is interrupted by one intron, and remarkably the promoter sequence, but not the 3′ untranslated sequence, is highly homologous to that of the corresponding gene. This situation appears to be unique with respect to the promoter regions of corresponding genes in related species of filamentous fungi.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-3-533
1996-03-01
2021-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/3/mic-142-3-533.html?itemId=/content/journal/micro/10.1099/13500872-142-3-533&mimeType=html&fmt=ahah

References

  1. Appleyard V. C. L., Unkles S. E., Legg M., Kinghorn J. R. 1995; Secondary metabolite production displayed. Mol & Gen Genet 247:338–342
    [Google Scholar]
  2. Avalos J., Casadesus J., Cerda-Olmedo E. 1994; Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N-nitro-N-nitrosoguanidine. Appi Environ Microbiol 49:187–191
    [Google Scholar]
  3. Banks G. R., Shelton P. A., Kanuga N., Holden D. W., Spanos A. 1993; The Ustilago maydis nari gene encoding nitrate reductase activity: structural and transcriptional regulation. Gene 131:69–78
    [Google Scholar]
  4. Bearder J. R. 1983; In vivo diterpenoid biosynthesis in Gibberella fujikuroi: the pathway after ent-kaurene. The Biochemistry and Physiology of Gibberellins251–387 Crozier A. New York: Praeger Publications;
    [Google Scholar]
  5. Brody H., Carbon J. 1989; Electrophoretic karyotype of Aspergillus nidulans . Proc Natl Acad SciUSA 866260–6263
    [Google Scholar]
  6. Brückner B., Blechschmidt D. 1991a; The gibberellin fermentation. Grit Rev Biotechnol 11:163–192
    [Google Scholar]
  7. Brückner B., Blechschmidt D. 1991b; Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi . Appl Microbiol Biotechnol 35:646–650
    [Google Scholar]
  8. Brückner B., Unkles S. E., Weltring K.-M., Kinghorn J. R. 1992; Transformation of Gibberella fujikuroi: effect of the Aspergillus nidulans AMAI sequence on frequency and integration. Curr Genet 22:314–316
    [Google Scholar]
  9. Bu’Lock J. D., Detroy R. W., Hostalek Z., Munim-al-Shakarchi A. 1974; Regulation of secondary metabolite biosynthesis in Gibberella fujikuroi . Trans Br Mycol Soc 62:377–389
    [Google Scholar]
  10. Burger G., Strauss J., Scazzocchio C., Lang B. 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]
  11. Campbell W. H., Kinghorn J. R. 1990; Functional domains of assimilatory nitrate and nitrite reductases. Trends Biochem Sci 15:315–319
    [Google Scholar]
  12. Candau R., Avalos J., Cerda-Olmedo E. 1991; Gibberellins and carotenoids in the wild type and mutants of Gibberella fujikuroi . Appl Environ Microbiol 57:3378–3382
    [Google Scholar]
  13. Candau R., Avalos J., Cerda-Olmedo E. 1992; Regulation of gibberellin biosynthesis in Gibberella fujikuroi . Plant Physiol 100:1184–1188
    [Google Scholar]
  14. Cove D. 1976; Chlorate toxicity in Aspergillus nidulans: the selection and characterisation of chlorate-resistant mutants. Heredity 36:191–203
    [Google Scholar]
  15. Diolez A., Langin T., Gerlinger C., Brygoo Y., Daboussi M.-J. 1993; The nia gene of Fusarium oxysporum : isolation, sequence and development of a homologous transformation system. Gene 131:61–67
    [Google Scholar]
  16. Fu Y.-H., Marzluf G. A. 1990; nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa encodes a sequence-specific DNA-binding protein. Proc Natl Acad SciUSA 875331–5335
    [Google Scholar]
  17. Fu Y.-H., Kneessi J. Y., Marzluf G. A. 1989; Isolation of nit-4, the minor regulatory gene which mediates nitrate induction in Neurospora crassa . J Bacteriol 171:4067–4070
    [Google Scholar]
  18. Hedden P., MacMillans J., Phinnel B. O. 1978; The metabolism of the gibberellins. Annu Rev Plant Physiol 29:149–150
    [Google Scholar]
  19. Johnstone I. L., MacCabe P. C., Greaves P., Cole G. E., Brow M. A. D., Gurr S. J., Unkles S. E., Clutterbuck A. J., Kinghorn J. R., Innis M. A. 1990; Isolation and characterisation of the crnA–niiA–niaD gene cluster for nitrate assimilation in Aspergillus nidulans . Gene 90:181–192
    [Google Scholar]
  20. Klittich C. J. R., Leslie J. F. 1988; Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi). Genetics 118:417–424
    [Google Scholar]
  21. Kudla B., Caddick M. X., Langdon T., Martinez-Rossi N. M., Bennett C. F., Sibley S., Davies R. W., Arst H. N. 1990; The regulatory gene are A mediating nitrogen metabolite repression in Aspergillus nidulans Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–1364
    [Google Scholar]
  22. Okamoto P. M., Fu Y.-H., Marzluf G. A. 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]
  23. Pitel D. W., Vining L. C., Arsenault G. P. 1971a; Improved methods for preparing pure gibberellins from cultures of Gibberella fujikuroi Isolation by adsorption or partition chromatography on silicic acid and by partition chromatography on Sephadex columns. Can J Biochem 49:185–193
    [Google Scholar]
  24. Pitel D. W., Vining L. C., Arsenault G. P. 1971b; Biosynthesis of gibberellins in Gibberella fujikuroi The sequence after gibberellin GA4. Can J Biochem 49:194–200
    [Google Scholar]
  25. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  26. Sanchez-Fernandez R., Unkles S. E., Campbell E. I., Macro J. A., Cerda-Olmedo E., Kinghorn J. R. 1991; Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus nidulans niaD gene encoding nitrate reductase. Mol & Gen Genet 225:231–233
    [Google Scholar]
  27. Smith C. L., Matsumoto T., Niwa O., Klco S., Fan J.-B., Yanagida M., Cantor C. R. 1987; An electrophoretic karyotype for Schizosaccharomyes pombe by pulsed field gel electrophoresis. Nucleic Acids Res 15:4481–4489
    [Google Scholar]
  28. Unkles S. E., Campbell E. I., Punt P. J., Hawker K. L, Contreras R., Hawkins A. R., van den Hondel C. A. M. J. J., Kinghorn J. R. 1992; The Aspergillus niger niaD gene encoding nitrate reductase : upstream nucleotide and amino acid comparisons. Gene 111:149–155
    [Google Scholar]
  29. Vass R. C., Jeffries E. G. 1979; Gibberellic acid. Economic Microbiology: Secondary Products of Metabolism421–435 Rose A. H. New York: Academic Press;
    [Google Scholar]
  30. Williams R. S. B., Davis M. A., Howlett B. J. 1994; Nitrate reductase of the ascomycetous fungus, Leptospbaeria maculans : gene sequence and chromosomal location. Mol & Gen Genet 244:1–8
    [Google Scholar]
  31. Xu J.-R., Yan K., Dickman M. B., Leslie J. F. 1995; Electro-phoretic karyotypes distinguish the biological species of Gibberella fujikuroi (Fusarium section liseola). Mol Plant-Microbe Interact 8:74–84
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-3-533
Loading
/content/journal/micro/10.1099/13500872-142-3-533
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error