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. 1994 Nov 11;22(22):4798–4805. doi: 10.1093/nar/22.22.4798

Sequence organization of the Acanthamoeba rRNA intergenic spacer: identification of transcriptional enhancers.

Q Yang 1, M G Zwick 1, M R Paule 1
PMCID: PMC308533  PMID: 7984432

Abstract

The primary sequence of the entire 2330 bp intergenic spacer of the A.castellanii ribosomal RNA gene was determined. Repeated sequence elements averaging 140 bp were identified and found to bind a protein required for optimum initiation at the core promoter. These repeated elements were shown to stimulate rRNA transcription by RNA polymerase I in vitro. The repeats inhibited transcription when placed in trans, and stimulated transcription when in cis, in either orientation, but only when upstream of the core promoter. Thus, these repeated elements have characteristics similar to polymerase I enhancers found in higher eukaryotes. The number of rRNA repeats in Acanthamoeba cells was determined to be 24 per haploid genome, the lowest number so far identified in any eukaryote. However, because Acanthamoeba is polyploid, each cell contains approximately 600 rRNA genes.

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Selected References

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

  1. Bach R., Grummt I., Allet B. The nucleotide sequence of the initiation region of the ribosomal transcription unit from mouse. Nucleic Acids Res. 1981 Apr 10;9(7):1559–1569. doi: 10.1093/nar/9.7.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bateman E., Iida C. T., Kownin P., Paule M. R. Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8004–8008. doi: 10.1073/pnas.82.23.8004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bateman E., Paule M. R. Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates. Mol Cell Biol. 1988 May;8(5):1940–1946. doi: 10.1128/mcb.8.5.1940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braaten D. C., Thomas J. R., Little R. D., Dickson K. R., Goldberg I., Schlessinger D., Ciccodicola A., D'Urso M. Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes. Nucleic Acids Res. 1988 Feb 11;16(3):865–881. doi: 10.1093/nar/16.3.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breslauer K. J., Frank R., Blöcker H., Marky L. A. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. doi: 10.1073/pnas.83.11.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Busby S. J., Reeder R. H. Spacer sequences regulate transcription of ribosomal gene plasmids injected into Xenopus embryos. Cell. 1983 Oct;34(3):989–996. doi: 10.1016/0092-8674(83)90556-1. [DOI] [PubMed] [Google Scholar]
  7. Cassidy B. G., Yang-Yen H. F., Rothblum L. I. Transcriptional role for the nontranscribed spacer of rat ribosomal DNA. Mol Cell Biol. 1986 Aug;6(8):2766–2773. doi: 10.1128/mcb.6.8.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. D'Alessio J. M., Harris G. H., Perna P. J., Paule M. R. Ribosomal ribonucleic acid repeat unit of Acanthamoeba castellanii: cloning and restriction endonuclease map. Biochemistry. 1981 Jun 23;20(13):3822–3827. doi: 10.1021/bi00516a024. [DOI] [PubMed] [Google Scholar]
  9. Dickson K. R., Braaten D. C., Schlessinger D. Human ribosomal DNA: conserved sequence elements in a 4.3-kb region downstream from the transcription unit. Gene. 1989 Dec 7;84(1):197–200. doi: 10.1016/0378-1119(89)90156-x. [DOI] [PubMed] [Google Scholar]
  10. Dixit A., Garg L. C., Chao W., Jacob S. T. An enhancer element in the far upstream spacer region of rat ribosomal RNA gene. J Biol Chem. 1987 Aug 25;262(24):11616–11622. [PubMed] [Google Scholar]
  11. Elion E. A., Warner J. R. An RNA polymerase I enhancer in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jun;6(6):2089–2097. doi: 10.1128/mcb.6.6.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elion E. A., Warner J. R. The major promoter element of rRNA transcription in yeast lies 2 kb upstream. Cell. 1984 Dec;39(3 Pt 2):663–673. doi: 10.1016/0092-8674(84)90473-2. [DOI] [PubMed] [Google Scholar]
  13. Fyrberg E. A., Kindle K. L., Davidson N., Kindle K. L. The actin genes of Drosophila: a dispersed multigene family. Cell. 1980 Feb;19(2):365–378. doi: 10.1016/0092-8674(80)90511-5. [DOI] [PubMed] [Google Scholar]
  14. Ghosh A. K., Hoff C. M., Jacob S. T. Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF. Gene. 1993 Mar 30;125(2):217–222. doi: 10.1016/0378-1119(93)90332-w. [DOI] [PubMed] [Google Scholar]
  15. Grimaldi G., Di Nocera P. P. Multiple repeated units in Drosophila melanogaster ribosomal DNA spacer stimulate rRNA precursor transcription. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5502–5506. doi: 10.1073/pnas.85.15.5502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grimaldi G., Fiorentini P., Di Nocera P. P. Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster. Mol Cell Biol. 1990 Sep;10(9):4667–4677. doi: 10.1128/mcb.10.9.4667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gruendler P., Unfried I., Pascher K., Schweizer D. rDNA intergenic region from Arabidopsis thaliana. Structural analysis, intraspecific variation and functional implications. J Mol Biol. 1991 Oct 20;221(4):1209–1222. doi: 10.1016/0022-2836(91)90929-z. [DOI] [PubMed] [Google Scholar]
  18. Gruss P., Dhar R., Khoury G. Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc Natl Acad Sci U S A. 1981 Feb;78(2):943–947. doi: 10.1073/pnas.78.2.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoff C. M., Jacob S. T. Characterization of the factor E1BF from a rat hepatoma that modulates ribosomal RNA gene transcription and its relationship to the human Ku autoantigen. Biochem Biophys Res Commun. 1993 Feb 15;190(3):747–753. doi: 10.1006/bbrc.1993.1112. [DOI] [PubMed] [Google Scholar]
  20. Iida C. T., Kownin P., Paule M. R. Ribosomal RNA transcription: proteins and DNA sequences involved in preinitiation complex formation. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1668–1672. doi: 10.1073/pnas.82.6.1668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Imperiale M. J., Feldman L. T., Nevins J. R. Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell. 1983 Nov;35(1):127–136. doi: 10.1016/0092-8674(83)90215-5. [DOI] [PubMed] [Google Scholar]
  22. Jacob S. T., Zhang J., Garg L. C., Book C. B. Multiple functional enhancer motifs of rat ribosomal gene. 1991 May 29-Jun 12Mol Cell Biochem. 104(1-2):155–162. doi: 10.1007/BF00229815. [DOI] [PubMed] [Google Scholar]
  23. Johnson S. P., Warner J. R. Unusual enhancer function in yeast rRNA transcription. Mol Cell Biol. 1989 Nov;9(11):4986–4993. doi: 10.1128/mcb.9.11.4986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jongstra J., Reudelhuber T. L., Oudet P., Benoist C., Chae C. B., Jeltsch J. M., Mathis D. J., Chambon P. Induction of altered chromatin structures by simian virus 40 enhancer and promoter elements. Nature. 1984 Feb 23;307(5953):708–714. doi: 10.1038/307708a0. [DOI] [PubMed] [Google Scholar]
  25. Kownin P., Bateman E., Paule M. R. Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I. Mol Cell Biol. 1988 Feb;8(2):747–753. doi: 10.1128/mcb.8.2.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kownin P., Bateman E., Paule M. R. Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent. Cell. 1987 Aug 28;50(5):693–699. doi: 10.1016/0092-8674(87)90327-8. [DOI] [PubMed] [Google Scholar]
  27. Kownin P., Iida C. T., Brown-Shimer S., Paule M. R. The ribosomal RNA promoter of Acanthamoeba castellanii determined by transcription in a cell-free system. Nucleic Acids Res. 1985 Sep 11;13(17):6237–6248. doi: 10.1093/nar/13.17.6237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kuhn A., Deppert U., Grummt I. A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7527–7531. doi: 10.1073/pnas.87.19.7527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kuhn A., Grummt I. Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7340–7344. doi: 10.1073/pnas.89.16.7340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. La Volpe A., Simeone A., D'Esposito M., Scotto L., Fidanza V., de Falco A., Boncinelli E. Molecular analysis of the heterogeneity region of the human ribosomal spacer. J Mol Biol. 1985 May 25;183(2):213–223. doi: 10.1016/0022-2836(85)90214-1. [DOI] [PubMed] [Google Scholar]
  31. Labhart P., Reeder R. H. DNA sequences for typical ribosomal gene spacers from Xenopus laevis and Xenopus borealis. Nucleic Acids Res. 1987 Apr 24;15(8):3623–3624. doi: 10.1093/nar/15.8.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Labhart P., Reeder R. H. Enhancer-like properties of the 60/81 bp elements in the ribosomal gene spacer of Xenopus laevis. Cell. 1984 May;37(1):285–289. doi: 10.1016/0092-8674(84)90324-6. [DOI] [PubMed] [Google Scholar]
  33. Levinson B., Khoury G., Vande Woude G., Gruss P. Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus. Nature. 1982 Feb 18;295(5850):568–572. doi: 10.1038/295568a0. [DOI] [PubMed] [Google Scholar]
  34. Long E. O., Dawid I. B. Repeated genes in eukaryotes. Annu Rev Biochem. 1980;49:727–764. doi: 10.1146/annurev.bi.49.070180.003455. [DOI] [PubMed] [Google Scholar]
  35. Moreau P., Hen R., Wasylyk B., Everett R., Gaub M. P., Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 1981 Nov 25;9(22):6047–6068. doi: 10.1093/nar/9.22.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moss T., Boseley P. G., Birnstiel M. L. More ribosomal spacer sequences from Xenopus laevis. Nucleic Acids Res. 1980 Feb 11;8(3):467–485. doi: 10.1093/nar/8.3.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pape L. K., Windle J. J., Mougey E. B., Sollner-Webb B. The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system. Mol Cell Biol. 1989 Nov;9(11):5093–5104. doi: 10.1128/mcb.9.11.5093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Paule M. R. Polymerase I transcription, termination, and processing. Gene Expr. 1993;3(1):1–9. [PMC free article] [PubMed] [Google Scholar]
  39. Perna P. J., Harris G. H., Iida C. T., Kownin P., Bugren S., Paule M. R. The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit. Gene Expr. 1992;2(1):71–78. [PMC free article] [PubMed] [Google Scholar]
  40. Pikaard C. S., Pape L. K., Henderson S. L., Ryan K., Paalman M. H., Lopata M. A., Reeder R. H., Sollner-Webb B. Enhancers for RNA polymerase I in mouse ribosomal DNA. Mol Cell Biol. 1990 Sep;10(9):4816–4825. doi: 10.1128/mcb.10.9.4816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Radebaugh C. A., Matthews J. L., Geiss G. K., Liu F., Wong J. M., Bateman E., Camier S., Sentenac A., Paule M. R. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors. Mol Cell Biol. 1994 Jan;14(1):597–605. doi: 10.1128/mcb.14.1.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Reeder R. H. Regulatory elements of the generic ribosomal gene. Curr Opin Cell Biol. 1989 Jun;1(3):466–474. doi: 10.1016/0955-0674(89)90007-0. [DOI] [PubMed] [Google Scholar]
  43. Reeder R. H., Roan J. G., Dunaway M. Spacer regulation of Xenopus ribosomal gene transcription: competition in oocytes. Cell. 1983 Dec;35(2 Pt 1):449–456. doi: 10.1016/0092-8674(83)90178-2. [DOI] [PubMed] [Google Scholar]
  44. Reeder R. H. rRNA synthesis in the nucleolus. Trends Genet. 1990 Dec;6(12):390–395. doi: 10.1016/0168-9525(90)90298-k. [DOI] [PubMed] [Google Scholar]
  45. Rychlik W., Rhoads R. E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 1989 Nov 11;17(21):8543–8551. doi: 10.1093/nar/17.21.8543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Schreier P. H., Cortese R. A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13. J Mol Biol. 1979 Mar 25;129(1):169–172. doi: 10.1016/0022-2836(79)90068-8. [DOI] [PubMed] [Google Scholar]
  49. Simeone A., La Volpe A., Boncinelli E. Nucleotide sequence of a complete ribosomal spacer of D. melanogaster. Nucleic Acids Res. 1985 Feb 25;13(4):1089–1101. doi: 10.1093/nar/13.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Skryabin K. G., Eldarov M. A., Larionov V. L., Bayev A. A., Klootwijk J., de Regt V. C., Veldman G. M., Planta R. J., Georgiev O. I., Hadjiolov A. A. Structure and function of the nontranscribed spacer regions of yeast rDNA. Nucleic Acids Res. 1984 Mar 26;12(6):2955–2968. doi: 10.1093/nar/12.6.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sylvester J. E., Petersen R., Schmickel R. D. Human ribosomal DNA: novel sequence organization in a 4.5-kb region upstream from the promoter. Gene. 1989 Dec 7;84(1):193–196. doi: 10.1016/0378-1119(89)90155-8. [DOI] [PubMed] [Google Scholar]
  52. Wasylyk B., Wasylyk C., Augereau P., Chambon P. The SV40 72 bp repeat preferentially potentiates transcription starting from proximal natural or substitute promoter elements. Cell. 1983 Feb;32(2):503–514. doi: 10.1016/0092-8674(83)90470-1. [DOI] [PubMed] [Google Scholar]
  53. Yang-Yen H. F., Rothblum L. I. Partial nucleotide sequence of a 3.4 kb fragment from the rat ribosomal DNA nontranscribed spacer. Nucleic Acids Res. 1986 Jul 11;14(13):5557–5557. doi: 10.1093/nar/14.13.5557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yang-Yen H. F., Rothblum L. I. Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro. Mol Cell Biol. 1988 Aug;8(8):3406–3414. doi: 10.1128/mcb.8.8.3406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yavachev L. P., Georgiev O. I., Braga E. A., Avdonina T. A., Bogomolova A. E., Zhurkin V. B., Nosikov V. V., Hadjiolov A. A. Nucleotide sequence analysis of the spacer regions flanking the rat rRNA transcription unit and identification of repetitive elements. Nucleic Acids Res. 1986 Mar 25;14(6):2799–2810. doi: 10.1093/nar/14.6.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]

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