Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Jul;7(7):2352–2359. doi: 10.1128/mcb.7.7.2352

Functional organization of the Aspergillus nidulans trpC promoter.

J E Hamer, W E Timberlake
PMCID: PMC365366  PMID: 3039345

Abstract

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

Full text

PDF
2352

Images in this article

2354Image
on p.2354
2356Image
on p.2356

Selected References

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

  1. Alton N. K., Buxton F., Patel V., Giles N. H., Vapnek D. 5'-Untranslated sequences of two structural genes in the qa gene cluster of Neurospora crassa. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1955–1959. doi: 10.1073/pnas.79.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  3. Boyce F. M., Anderson G. M., Rusk C. D., Freytag S. O. Human argininosuccinate synthetase minigenes are subject to arginine-mediated repression but not to trans induction. Mol Cell Biol. 1986 Apr;6(4):1244–1252. doi: 10.1128/mcb.6.4.1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Boylan M. T., Holland M. J., Timberlake W. E. Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome. Mol Cell Biol. 1986 Nov;6(11):3621–3625. doi: 10.1128/mcb.6.11.3621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Burke J. F. High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene. 1984 Oct;30(1-3):63–68. doi: 10.1016/0378-1119(84)90105-7. [DOI] [PubMed] [Google Scholar]
  8. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  9. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  10. Close T. J., Rodriguez R. L. Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene. 1982 Dec;20(2):305–316. doi: 10.1016/0378-1119(82)90048-8. [DOI] [PubMed] [Google Scholar]
  11. Fantes P. A., Roberts C. F. Beta-galactosidase activity and lactose utilization in Aspergillus nidulans. J Gen Microbiol. 1973 Aug;77(2):417–486. doi: 10.1099/00221287-77-2-417. [DOI] [PubMed] [Google Scholar]
  12. Goodbourn S., Burstein H., Maniatis T. The human beta-interferon gene enhancer is under negative control. Cell. 1986 May 23;45(4):601–610. doi: 10.1016/0092-8674(86)90292-8. [DOI] [PubMed] [Google Scholar]
  13. Grosschedl R., Birnstiel M. L. Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1432–1436. doi: 10.1073/pnas.77.3.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grosveld G. C., de Boer E., Shewmaker C. K., Flavell R. A. DNA sequences necessary for transcription of the rabbit beta-globin gene in vivo. Nature. 1982 Jan 14;295(5845):120–126. doi: 10.1038/295120a0. [DOI] [PubMed] [Google Scholar]
  15. Guarente L., Hoar E. Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box". Proc Natl Acad Sci U S A. 1984 Dec;81(24):7860–7864. doi: 10.1073/pnas.81.24.7860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hahn S., Hoar E. T., Guarente L. Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8562–8566. doi: 10.1073/pnas.82.24.8562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haltiner M., Kempe T., Tjian R. A novel strategy for constructing clustered point mutations. Nucleic Acids Res. 1985 Feb 11;13(3):1015–1025. doi: 10.1093/nar/13.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  19. Käfer E. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet. 1977;19:33–131. doi: 10.1016/s0065-2660(08)60245-x. [DOI] [PubMed] [Google Scholar]
  20. Käfer E. The anthranilate synthetase enzyme complex and the trifunctional trpC gene of Aspergillus. Can J Genet Cytol. 1977 Dec;19(4):723–738. doi: 10.1139/g77-079. [DOI] [PubMed] [Google Scholar]
  21. Legerton T. L., Yanofsky C. Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa. Gene. 1985;39(2-3):129–140. doi: 10.1016/0378-1119(85)90306-3. [DOI] [PubMed] [Google Scholar]
  22. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  23. McKnight G. L., Kato H., Upshall A., Parker M. D., Saari G., O'Hara P. J. Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. EMBO J. 1985 Aug;4(8):2093–2099. doi: 10.1002/j.1460-2075.1985.tb03897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McKnight G. L., O'Hara P. J., Parker M. L. Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns. Cell. 1986 Jul 4;46(1):143–147. doi: 10.1016/0092-8674(86)90868-8. [DOI] [PubMed] [Google Scholar]
  25. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  26. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  27. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  28. Miller B. L., Miller K. Y., Timberlake W. E. Direct and indirect gene replacements in Aspergillus nidulans. Mol Cell Biol. 1985 Jul;5(7):1714–1721. doi: 10.1128/mcb.5.7.1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mullaney E. J., Hamer J. E., Roberti K. A., Yelton M. M., Timberlake W. E. Primary structure of the trpC gene from Aspergillus nidulans. Mol Gen Genet. 1985;199(1):37–45. doi: 10.1007/BF00327506. [DOI] [PubMed] [Google Scholar]
  30. Myers R. M., Tilly K., Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science. 1986 May 2;232(4750):613–618. doi: 10.1126/science.3457470. [DOI] [PubMed] [Google Scholar]
  31. Nagawa F., Fink G. R. The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8557–8561. doi: 10.1073/pnas.82.24.8557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nunberg J. H., Meade J. H., Cole G., Lawyer F. C., McCabe P., Schweickart V., Tal R., Wittman V. P., Flatgaard J. E., Innis M. A. Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori. Mol Cell Biol. 1984 Nov;4(11):2306–2315. doi: 10.1128/mcb.4.11.2306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reynolds G. A., Basu S. K., Osborne T. F., Chin D. J., Gil G., Brown M. S., Goldstein J. L., Luskey K. L. HMG CoA reductase: a negatively regulated gene with unusual promoter and 5' untranslated regions. Cell. 1984 Aug;38(1):275–285. doi: 10.1016/0092-8674(84)90549-x. [DOI] [PubMed] [Google Scholar]
  34. Roberts C. F. Complementation analysis of the tryptophan pathway in Aspergillus nidulans. Genetics. 1967 Feb;55(2):233–239. doi: 10.1093/genetics/55.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Russell P., Nurse P. Schizosaccharomyces pombe and Saccharomyces cerevisiae: a look at yeasts divided. Cell. 1986 Jun 20;45(6):781–782. doi: 10.1016/0092-8674(86)90550-7. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schechtman M. G., Yanofsky C. Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli. J Mol Appl Genet. 1983;2(1):83–99. [PubMed] [Google Scholar]
  38. Struhl K. Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7865–7869. doi: 10.1073/pnas.81.24.7865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Timberlake W. E. Developmental gene regulation in Aspergillus nidulans. Dev Biol. 1980 Aug;78(2):497–510. doi: 10.1016/0012-1606(80)90349-8. [DOI] [PubMed] [Google Scholar]
  40. Tyler B. M., Geever R. F., Case M. E., Giles N. H. Cis-acting and trans-acting regulatory mutations define two types of promoters controlled by the qa-1F gene of Neurospora. Cell. 1984 Feb;36(2):493–502. doi: 10.1016/0092-8674(84)90242-3. [DOI] [PubMed] [Google Scholar]
  41. Upshall A., Gilbert T., Saari G., O'Hara P. J., Weglenski P., Berse B., Miller K., Timberlake W. E. Molecular analysis of the argB gene of Aspergillus nidulans. Mol Gen Genet. 1986 Aug;204(2):349–354. doi: 10.1007/BF00425521. [DOI] [PubMed] [Google Scholar]
  42. Valerio D., Duyvesteyn M. G., Dekker B. M., Weeda G., Berkvens T. M., van der Voorn L., van Ormondt H., van der Eb A. J. Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J. 1985 Feb;4(2):437–443. doi: 10.1002/j.1460-2075.1985.tb03648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yelton M. M., Hamer J. E., Timberlake W. E. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1470–1474. doi: 10.1073/pnas.81.5.1470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yelton M. M., Hamer J. E., de Souza E. R., Mullaney E. J., Timberlake W. E. Developmental regulation of the Aspergillus nidulans trpC gene. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7576–7580. doi: 10.1073/pnas.80.24.7576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yelton M. M., Timberlake W. E., Hondel C. A. A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus. Proc Natl Acad Sci U S A. 1985 Feb;82(3):834–838. doi: 10.1073/pnas.82.3.834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. van Gorcom R. F., Pouwels P. H., Goosen T., Visser J., van den Broek H. W., Hamer J. E., Timberlake W. E., van den Hondel C. A. Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans. Gene. 1985;40(1):99–106. doi: 10.1016/0378-1119(85)90028-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES