Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Dec;15(12):7117–7126. doi: 10.1128/mcb.15.12.7117

An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila.

D K Butler 1, L E Yasuda 1, M C Yao 1
PMCID: PMC230967  PMID: 8524279

Abstract

Large palindromic DNAs are found in a wide variety of eukaryotic cells. In Tetrahymena thermophila, a large palindrome is formed from a single rRNA gene (rDNA) during nuclear differentiation. We present evidence that a key step in the formation of the rDNA palindrome of T. thermophila involves homologous intramolecular recombination. Heteroduplex micronuclear rDNA molecules were constructed in vitro and microinjected into developing macronuclei, where they formed palindromes. Analysis of the resulting palindromes indicated that both strands of the microinjected rDNA are used to form the same palindrome. This study, together with a previous study (L. F. Yasuda and M.-C. Yao, Cell 67:505-516, 1991), is the first to define a molecular pathway of palindrome formation. The process is initiated by chromosome breakage at sites flanking the micronuclear rDNA. An intramolecular recombination reaction, guided by a pair of short inverted repeats located at the 5' end of the excised rDNA, covalently joins the two strands of micronuclear rDNA in a giant hairpin molecule. Bidirectional DNA replication converts the giant hairpin molecule to a palindrome. We suggest that the general features of this pathway are applicable to palindrome formation in other cell types.

Full Text

The Full Text of this article is available as a PDF (466.2 KB).

Selected References

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

  1. Austerberry C. F., Yao M. C. Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila. Mol Cell Biol. 1987 Jan;7(1):435–443. doi: 10.1128/mcb.7.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett R. J., Dunderdale H. J., West S. C. Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell. 1993 Sep 24;74(6):1021–1031. doi: 10.1016/0092-8674(93)90724-5. [DOI] [PubMed] [Google Scholar]
  3. Beverley S. M., Coderre J. A., Santi D. V., Schimke R. T. Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell. 1984 Sep;38(2):431–439. doi: 10.1016/0092-8674(84)90498-7. [DOI] [PubMed] [Google Scholar]
  4. Butler D. K. Ribosomal DNA is a site of chromosome breakage in aneuploid strains of Neurospora. Genetics. 1992 Jul;131(3):581–592. doi: 10.1093/genetics/131.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carroll D., Wright S. H., Wolff R. K., Grzesiuk E., Maryon E. B. Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes. Mol Cell Biol. 1986 Jun;6(6):2053–2061. doi: 10.1128/mcb.6.6.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S., Hassin D., Karby S., Lavi S. Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification. Mol Cell Biol. 1994 Dec;14(12):7782–7791. doi: 10.1128/mcb.14.12.7782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cotmore S. F., Tattersall P. An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication. EMBO J. 1994 Sep 1;13(17):4145–4152. doi: 10.1002/j.1460-2075.1994.tb06732.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engberg J., Andersson P., Leick V., Collins J. Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes. J Mol Biol. 1976 Jun 25;104(2):455–470. doi: 10.1016/0022-2836(76)90281-3. [DOI] [PubMed] [Google Scholar]
  9. Fishman-Lobell J., Rudin N., Haber J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992 Mar;12(3):1292–1303. doi: 10.1128/mcb.12.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ford M., Fried M. Large inverted duplications are associated with gene amplification. Cell. 1986 May 9;45(3):425–430. doi: 10.1016/0092-8674(86)90328-4. [DOI] [PubMed] [Google Scholar]
  11. Fried M., Feo S., Heard E. The role of inverted duplication in the generation of gene amplification in mammalian cells. Biochim Biophys Acta. 1991 Oct 8;1090(2):143–155. doi: 10.1016/0167-4781(91)90095-4. [DOI] [PubMed] [Google Scholar]
  12. Godiska R., James C., Yao M. C. A distant 10-bp sequence specifies the boundaries of a programmed DNA deletion in Tetrahymena. Genes Dev. 1993 Dec;7(12A):2357–2365. doi: 10.1101/gad.7.12a.2357. [DOI] [PubMed] [Google Scholar]
  13. Haber J. E., Thorburn P. C. Healing of broken linear dicentric chromosomes in yeast. Genetics. 1984 Feb;106(2):207–226. doi: 10.1093/genetics/106.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hyde H., Davies A. A., Benson F. E., West S. C. Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase. J Biol Chem. 1994 Feb 18;269(7):5202–5209. [PubMed] [Google Scholar]
  15. Hyrien O., Debatisse M., Buttin G., de Saint Vincent B. R. The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification. EMBO J. 1988 Feb;7(2):407–417. doi: 10.1002/j.1460-2075.1988.tb02828.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jäger D., Philippsen P. Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion. EMBO J. 1989 Jan;8(1):247–254. doi: 10.1002/j.1460-2075.1989.tb03370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karrer K. M., Gall J. G. The macronuclear ribosomal DNA of Tetrahymena pyriformis is a palindrome. J Mol Biol. 1976 Jun 25;104(2):421–453. doi: 10.1016/0022-2836(76)90280-1. [DOI] [PubMed] [Google Scholar]
  18. Koshland D., Rutledge L., Fitzgerald-Hayes M., Hartwell L. H. A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae. Cell. 1987 Mar 13;48(5):801–812. doi: 10.1016/0092-8674(87)90077-8. [DOI] [PubMed] [Google Scholar]
  19. Kramer K. M., Haber J. E. New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats. Genes Dev. 1993 Dec;7(12A):2345–2356. doi: 10.1101/gad.7.12a.2345. [DOI] [PubMed] [Google Scholar]
  20. Lin F. L., Sperle K., Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol. 1984 Jun;4(6):1020–1034. doi: 10.1128/mcb.4.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ma C., Looney J. E., Leu T. H., Hamlin J. L. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line. Mol Cell Biol. 1988 Jun;8(6):2316–2327. doi: 10.1128/mcb.8.6.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ma C., Martin S., Trask B., Hamlin J. L. Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells. Genes Dev. 1993 Apr;7(4):605–620. doi: 10.1101/gad.7.4.605. [DOI] [PubMed] [Google Scholar]
  23. Matsumoto T., Fukui K., Niwa O., Sugawara N., Szostak J. W., Yanagida M. Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe. Mol Cell Biol. 1987 Dec;7(12):4424–4430. doi: 10.1128/mcb.7.12.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McClintock B. The Stability of Broken Ends of Chromosomes in Zea Mays. Genetics. 1941 Mar;26(2):234–282. doi: 10.1093/genetics/26.2.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Merchlinsky M., Moss B. Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions. Cell. 1986 Jun 20;45(6):879–884. doi: 10.1016/0092-8674(86)90562-3. [DOI] [PubMed] [Google Scholar]
  26. Merriam E. V., Bruns P. J. Phenotypic assortment in Tetrahymena thermophila: assortment kinetics of antibiotic-resistance markers, tsA, death, and the highly amplified rDNA locus. Genetics. 1988 Oct;120(2):389–395. doi: 10.1093/genetics/120.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ouellette M., Hettema E., Wüst D., Fase-Fowler F., Borst P. Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania. EMBO J. 1991 Apr;10(4):1009–1016. doi: 10.1002/j.1460-2075.1991.tb08035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pan W. C., Orias E., Flacks M., Blackburn E. H. Allele-specific, selective amplification of a ribosomal RNA gene in Tetrahymena thermophila. Cell. 1982 Mar;28(3):595–604. doi: 10.1016/0092-8674(82)90214-8. [DOI] [PubMed] [Google Scholar]
  30. Passananti C., Davies B., Ford M., Fried M. Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplification. EMBO J. 1987 Jun;6(6):1697–1703. doi: 10.1002/j.1460-2075.1987.tb02420.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pologe L. G., Ravetch J. V. Large deletions result from breakage and healing of P. falciparum chromosomes. Cell. 1988 Dec 2;55(5):869–874. doi: 10.1016/0092-8674(88)90142-0. [DOI] [PubMed] [Google Scholar]
  32. Roth D. B., Menetski J. P., Nakajima P. B., Bosma M. J., Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992 Sep 18;70(6):983–991. doi: 10.1016/0092-8674(92)90248-b. [DOI] [PubMed] [Google Scholar]
  33. Ruiz J. C., Wahl G. M. Formation of an inverted duplication can be an initial step in gene amplification. Mol Cell Biol. 1988 Oct;8(10):4302–4313. doi: 10.1128/mcb.8.10.4302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Snyder R. O., Samulski R. J., Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends. Cell. 1990 Jan 12;60(1):105–113. doi: 10.1016/0092-8674(90)90720-y. [DOI] [PubMed] [Google Scholar]
  35. Stark G. R., Debatisse M., Giulotto E., Wahl G. M. Recent progress in understanding mechanisms of mammalian DNA amplification. Cell. 1989 Jun 16;57(6):901–908. doi: 10.1016/0092-8674(89)90328-0. [DOI] [PubMed] [Google Scholar]
  36. Sweeney R., Yao M. C. Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila. EMBO J. 1989 Mar;8(3):933–938. doi: 10.1002/j.1460-2075.1989.tb03454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Symington L. S., Kolodner R. Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7247–7251. doi: 10.1073/pnas.82.21.7247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Toledo F., Le Roscouet D., Buttin G., Debatisse M. Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification. EMBO J. 1992 Jul;11(7):2665–2673. doi: 10.1002/j.1460-2075.1992.tb05332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tondravi M. M., Yao M. C. Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4369–4373. doi: 10.1073/pnas.83.12.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Walton J. D., Paquin C. E., Kaneko K., Williamson V. M. Resistance to antimycin A in yeast by amplification of ADH4 on a linear, 42 kb palindromic plasmid. Cell. 1986 Sep 12;46(6):857–863. doi: 10.1016/0092-8674(86)90067-x. [DOI] [PubMed] [Google Scholar]
  41. West S. C., Körner A. Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6445–6449. doi: 10.1073/pnas.82.19.6445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wilkie A. O., Lamb J., Harris P. C., Finney R. D., Higgs D. R. A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n. Nature. 1990 Aug 30;346(6287):868–871. doi: 10.1038/346868a0. [DOI] [PubMed] [Google Scholar]
  43. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  44. Yao M. C., Yao C. H. Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila. Mol Cell Biol. 1989 Mar;9(3):1092–1099. doi: 10.1128/mcb.9.3.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yao M. C., Yao C. H., Monks B. The controlling sequence for site-specific chromosome breakage in Tetrahymena. Cell. 1990 Nov 16;63(4):763–772. doi: 10.1016/0092-8674(90)90142-2. [DOI] [PubMed] [Google Scholar]
  46. Yasuda L. F., Yao M. C. Short inverted repeats at a free end signal large palindromic DNA formation in Tetrahymena. Cell. 1991 Nov 1;67(3):505–516. doi: 10.1016/0092-8674(91)90525-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES