Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5688–5699. doi: 10.1128/mcb.15.10.5688

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

P J Punt 1, J Strauss 1, R Smit 1, J R Kinghorn 1, C A van den Hondel 1, C Scazzocchio 1
PMCID: PMC230819  PMID: 7565720

Abstract

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

Full Text

The Full Text of this article is available as a PDF (723.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arst H. N., Jr, Cove D. J. Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet. 1973 Nov 2;126(2):111–141. doi: 10.1007/BF00330988. [DOI] [PubMed] [Google Scholar]
  2. Arst H. N., Jr, Jones S. A., Bailey C. R. A method for the selection of deletion mutations in the L-proline catabolism gene cluster of Aspergillus nidulans. Genet Res. 1981 Oct;38(2):171–195. doi: 10.1017/s0016672300020516. [DOI] [PubMed] [Google Scholar]
  3. Baum J. A., Geever R., Giles N. H. Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol Cell Biol. 1987 Mar;7(3):1256–1266. doi: 10.1128/mcb.7.3.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brownlee A. G., Arst H. N., Jr Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster. J Bacteriol. 1983 Sep;155(3):1138–1146. doi: 10.1128/jb.155.3.1138-1146.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burger G., Strauss J., Scazzocchio C., Lang B. F. 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. 1991 Nov;11(11):5746–5755. doi: 10.1128/mcb.11.11.5746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burger G., Tilburn J., Scazzocchio C. Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans. Mol Cell Biol. 1991 Feb;11(2):795–802. doi: 10.1128/mcb.11.2.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. An amino-terminal fragment of GAL4 binds DNA as a dimer. J Mol Biol. 1989 Oct 5;209(3):423–432. doi: 10.1016/0022-2836(89)90007-7. [DOI] [PubMed] [Google Scholar]
  8. Cove D. J. Evidence for a near limiting intracellular concentration of a regulator substance. Nature. 1969 Oct 18;224(5216):272–273. doi: 10.1038/224272b0. [DOI] [PubMed] [Google Scholar]
  9. Cove D. J. Genetic studies of nitrate assimilation in Aspergillus nidulans. Biol Rev Camb Philos Soc. 1979 Aug;54(3):291–327. doi: 10.1111/j.1469-185x.1979.tb01014.x. [DOI] [PubMed] [Google Scholar]
  10. Cove D. J., Pateman J. A. Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. J Bacteriol. 1969 Mar;97(3):1374–1378. doi: 10.1128/jb.97.3.1374-1378.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cubero B., Scazzocchio C. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. EMBO J. 1994 Jan 15;13(2):407–415. doi: 10.1002/j.1460-2075.1994.tb06275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Daboussi M. J., Langin T., Deschamps F., Brygoo Y., Scazzocchio C., Burger G. Heterologous expression of the Aspergillus nidulans regulatory gene nirA in Fusarium oxysporum. Gene. 1991 Dec 20;109(1):155–160. doi: 10.1016/0378-1119(91)90602-8. [DOI] [PubMed] [Google Scholar]
  13. De Rijcke M., Seneca S., Punyammalee B., Glansdorff N., Crabeel M. Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine. Mol Cell Biol. 1992 Jan;12(1):68–81. doi: 10.1128/mcb.12.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  15. Diolez A., Langin T., Gerlinger C., Brygoo Y., Daboussi M. J. The nia gene of Fusarium oxysporum: isolation, sequence and development of a homologous transformation system. Gene. 1993 Sep 6;131(1):61–67. doi: 10.1016/0378-1119(93)90669-t. [DOI] [PubMed] [Google Scholar]
  16. Exley G. E., Colandene J. D., Garrett R. H. Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa. J Bacteriol. 1993 Apr;175(8):2379–2392. doi: 10.1128/jb.175.8.2379-2392.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol. 1988 Jul;8(7):2690–2697. doi: 10.1128/mcb.8.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fu Y. H., Feng B., Evans S., Marzluf G. A. Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora. Mol Microbiol. 1995 Mar;15(5):935–942. doi: 10.1111/j.1365-2958.1995.tb02362.x. [DOI] [PubMed] [Google Scholar]
  19. Fu Y. H., Marzluf G. A. nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5331–5335. doi: 10.1073/pnas.87.14.5331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geever R. F., Huiet L., Baum J. A., Tyler B. M., Patel V. B., Rutledge B. J., Case M. E., Giles N. H. DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa. J Mol Biol. 1989 May 5;207(1):15–34. doi: 10.1016/0022-2836(89)90438-5. [DOI] [PubMed] [Google Scholar]
  21. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  22. Halvorsen Y. D., Nandabalan K., Dickson R. C. Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger. Mol Cell Biol. 1991 Apr;11(4):1777–1784. doi: 10.1128/mcb.11.4.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hawker K. L., Montague P., Kinghorn J. R. Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation. Mol Gen Genet. 1992 Feb;231(3):485–488. doi: 10.1007/BF00292720. [DOI] [PubMed] [Google Scholar]
  24. Hawker K. L., Montague P., Marzluf G. A., Kinghorn J. R. Heterologous expression and regulation of the Neurospora crassa nit-4 pathway-specific regulatory gene for nitrate assimilation in Aspergillus nidulans. Gene. 1991 Apr;100:237–240. doi: 10.1016/0378-1119(91)90373-j. [DOI] [PubMed] [Google Scholar]
  25. Hawkins A. R., Lamb H. K., Roberts C. F. Control of metabolic flux in the shikimate and quinate pathways. Prog Ind Microbiol. 1994;29:195–220. [PubMed] [Google Scholar]
  26. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  27. Hull E. P., Green P. M., Arst H. N., Jr, Scazzocchio C. Cloning and physical characterization of the L-proline catabolism gene cluster of Aspergillus nidulans. Mol Microbiol. 1989 Apr;3(4):553–559. doi: 10.1111/j.1365-2958.1989.tb00201.x. [DOI] [PubMed] [Google Scholar]
  28. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Johnstone I. L., McCabe P. C., Greaves P., Gurr S. J., Cole G. E., Brow M. A., Unkles S. E., Clutterbuck A. J., Kinghorn J. R., Innis M. A. Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene. 1990 Jun 15;90(2):181–192. doi: 10.1016/0378-1119(90)90178-t. [DOI] [PubMed] [Google Scholar]
  30. Kudla B., Caddick M. X., Langdon T., Martinez-Rossi N. M., Bennett C. F., Sibley S., Davies R. W., Arst H. N., Jr The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J. 1990 May;9(5):1355–1364. doi: 10.1002/j.1460-2075.1990.tb08250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kulmburg P., Judewicz N., Mathieu M., Lenouvel F., Sequeval D., Felenbok B. Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans. J Biol Chem. 1992 Oct 15;267(29):21146–21153. [PubMed] [Google Scholar]
  32. Kulmburg P., Sequeval D., Lenouvel F., Mathieu M., Felenbok B. Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans. Mol Cell Biol. 1992 May;12(5):1932–1939. doi: 10.1128/mcb.12.5.1932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Malardier L., Daboussi M. J., Julien J., Roussel F., Scazzocchio C., Brygoo Y. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene. 1989 May 15;78(1):147–156. doi: 10.1016/0378-1119(89)90322-3. [DOI] [PubMed] [Google Scholar]
  34. Marmorstein R., Carey M., Ptashne M., Harrison S. C. DNA recognition by GAL4: structure of a protein-DNA complex. Nature. 1992 Apr 2;356(6368):408–414. doi: 10.1038/356408a0. [DOI] [PubMed] [Google Scholar]
  35. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Okamoto P. M., Fu Y. H., Marzluf G. A. Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet. 1991 Jun;227(2):213–223. doi: 10.1007/BF00259673. [DOI] [PubMed] [Google Scholar]
  37. PONTECORVO G., ROPER J. A., HEMMONS L. M., MACDONALD K. D., BUFTON A. W. J. The genetics of Aspergillus nidulans. Adv Genet. 1953;5:141–238. doi: 10.1016/s0065-2660(08)60408-3. [DOI] [PubMed] [Google Scholar]
  38. Pateman J. A., Cove D. J. Regulation of nitrate reduction in Aspergillus nidulans. Nature. 1967 Sep 16;215(5107):1234–1237. doi: 10.1038/2151234a0. [DOI] [PubMed] [Google Scholar]
  39. Pateman J. A., Doy C. H., Olsen J. E., Norris U., Creaser E. H., Hynes M. Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans. Proc R Soc Lond B Biol Sci. 1983 Feb 22;217(1208):243–264. doi: 10.1098/rspb.1983.0009. [DOI] [PubMed] [Google Scholar]
  40. Punt P. J., Greaves P. A., Kuyvenhoven A., van Deutekom J. C., Kinghorn J. R., Pouwels P. H., van den Hondel C. A. A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi. Gene. 1991 Jul 31;104(1):119–122. doi: 10.1016/0378-1119(91)90476-r. [DOI] [PubMed] [Google Scholar]
  41. Punt P. J., Kramer C., Kuyvenhoven A., Pouwels P. H., van den Hondel C. A. An upstream activating sequence from the Aspergillus nidulans gpdA gene. Gene. 1992 Oct 12;120(1):67–73. doi: 10.1016/0378-1119(92)90010-m. [DOI] [PubMed] [Google Scholar]
  42. Punt P. J., van den Hondel C. A. Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol. 1992;216:447–457. doi: 10.1016/0076-6879(92)16041-h. [DOI] [PubMed] [Google Scholar]
  43. Rand K. N., Arst H. N., Jr Mutations in nirA gene of Aspergillus nidulans and nitrogen metabolism. Nature. 1978 Apr 20;272(5655):732–734. doi: 10.1038/272732a0. [DOI] [PubMed] [Google Scholar]
  44. Roy A., Exinger F., Losson R. cis- and trans-acting regulatory elements of the yeast URA3 promoter. Mol Cell Biol. 1990 Oct;10(10):5257–5270. doi: 10.1128/mcb.10.10.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siddiqui A. H., Brandriss M. C. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences. Mol Cell Biol. 1989 Nov;9(11):4706–4712. doi: 10.1128/mcb.9.11.4706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sophianopoulou V., Suárez T., Diallinas G., Scazzocchio C. Operator derepressed mutations in the proline utilisation gene cluster of Aspergillus nidulans. Mol Gen Genet. 1993 Jan;236(2-3):209–213. doi: 10.1007/BF00277114. [DOI] [PubMed] [Google Scholar]
  47. Suárez T., de Queiroz M. V., Oestreicher N., Scazzocchio C. The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae. EMBO J. 1995 Apr 3;14(7):1453–1467. doi: 10.1002/j.1460-2075.1995.tb07132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tomsett A. B., Cove D. J. Deletion mapping of the niiA niaD gene region of Aspergillus nidulans. Genet Res. 1979 Aug;34(1):19–32. doi: 10.1017/s001667230001925x. [DOI] [PubMed] [Google Scholar]
  49. Unkles S. E., Campbell E. I., Punt P. J., Hawker K. L., Contreras R., Hawkins A. R., Van den Hondel C. A., Kinghorn J. R. The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons. Gene. 1992 Feb 15;111(2):149–155. doi: 10.1016/0378-1119(92)90682-f. [DOI] [PubMed] [Google Scholar]
  50. Unkles S. E., Hawker K. L., Grieve C., Campbell E. I., Montague P., Kinghorn J. R. crnA encodes a nitrate transporter in Aspergillus nidulans. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):204–208. doi: 10.1073/pnas.88.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. West R. W., Jr, Yocum R. R., Ptashne M. Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984 Nov;4(11):2467–2478. doi: 10.1128/mcb.4.11.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Williams R. S., Davis M. A., Howlett B. J. Nitrate reductase of the ascomycetous fungus, Leptosphaeria maculans: gene sequence and chromosomal location. Mol Gen Genet. 1994 Jul 8;244(1):1–8. doi: 10.1007/BF00280180. [DOI] [PubMed] [Google Scholar]
  53. Williams R. S., Davis M. A., Howlett B. J. The nitrate and nitrite reductase-encoding genes of Leptosphaeria maculans are closely linked and transcribed in the same direction. Gene. 1995 May 26;158(1):153–154. doi: 10.1016/0378-1119(95)00169-7. [DOI] [PubMed] [Google Scholar]
  54. Yuan G. F., Fu Y. H., Marzluf G. A. nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol Cell Biol. 1991 Nov;11(11):5735–5745. doi: 10.1128/mcb.11.11.5735. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES