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. 1988 Apr;8(4):1664–1669. doi: 10.1128/mcb.8.4.1664

Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

K M Karrer 1, M C Yao 1
PMCID: PMC363326  PMID: 3380092

Abstract

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

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Selected References

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

  1. Allitto B. A., Karrer K. M. A family of DNA sequences is reproducibly rearranged in the somatic nucleus of Tetrahymena. Nucleic Acids Res. 1986 Oct 24;14(20):8007–8025. doi: 10.1093/nar/14.20.8007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bird A. P. Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern. J Mol Biol. 1978 Jan 5;118(1):49–60. doi: 10.1016/0022-2836(78)90243-7. [DOI] [PubMed] [Google Scholar]
  4. Blackburn E. H., Pan W. C., Johnson C. C. Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila. Nucleic Acids Res. 1983 Aug 11;11(15):5131–5145. doi: 10.1093/nar/11.15.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bromberg S., Pratt K., Hattman S. Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila. J Bacteriol. 1982 May;150(2):993–996. doi: 10.1128/jb.150.2.993-996.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Budarf M. L., Blackburn E. H. Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes. J Biol Chem. 1986 Jan 5;261(1):363–369. [PubMed] [Google Scholar]
  7. Cartinhour S. W., Herrick G. A. Three different macronuclear DNAs in Oxytricha fallax share a common sequence block. Mol Cell Biol. 1984 May;4(5):931–938. doi: 10.1128/mcb.4.5.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cech T. R., Brehm S. L. Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia. Nucleic Acids Res. 1981 Jul 24;9(14):3531–3543. doi: 10.1093/nar/9.14.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Din N., Engberg J. Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution. J Mol Biol. 1979 Nov 5;134(3):555–574. doi: 10.1016/0022-2836(79)90367-x. [DOI] [PubMed] [Google Scholar]
  10. Gorovsky M. A., Hattman S., Pleger G. L. ( 6 N)methyl adenine in the nuclear DNA of a eucaryote, Tetrahymena pyriformis. J Cell Biol. 1973 Mar;56(3):697–701. doi: 10.1083/jcb.56.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorovsky M. A., Yao M. C., Keevert J. B., Pleger G. L. Isolation of micro- and macronuclei of Tetrahymena pyriformis. Methods Cell Biol. 1975;9(0):311–327. doi: 10.1016/s0091-679x(08)60080-1. [DOI] [PubMed] [Google Scholar]
  12. Gruenbaum Y., Cedar H., Razin A. Substrate and sequence specificity of a eukaryotic DNA methylase. Nature. 1982 Feb 18;295(5850):620–622. doi: 10.1038/295620a0. [DOI] [PubMed] [Google Scholar]
  13. Harrison G. S., Findly R. C., Karrer K. M. Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila. Mol Cell Biol. 1986 Jul;6(7):2364–2370. doi: 10.1128/mcb.6.7.2364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
  15. Keshet I., Lieman-Hurwitz J., Cedar H. DNA methylation affects the formation of active chromatin. Cell. 1986 Feb 28;44(4):535–543. doi: 10.1016/0092-8674(86)90263-1. [DOI] [PubMed] [Google Scholar]
  16. Larson D. D., Blackburn E. H., Yaeger P. C., Orias E. Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element. Cell. 1986 Oct 24;47(2):229–240. doi: 10.1016/0092-8674(86)90445-9. [DOI] [PubMed] [Google Scholar]
  17. Larson D. D., Spangler E. A., Blackburn E. H. Dynamics of telomere length variation in Tetrahymena thermophila. Cell. 1987 Jul 31;50(3):477–483. doi: 10.1016/0092-8674(87)90501-0. [DOI] [PubMed] [Google Scholar]
  18. Martindale D. W., Martindale H. M., Bruns P. J. Tetrahymena conjugation-induced genes: structure and organization in macro- and micronuclei. Nucleic Acids Res. 1986 Feb 11;14(3):1341–1354. doi: 10.1093/nar/14.3.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Orias E., Bruns P. J. Induction and isolation of mutants in Tetrahymena. Methods Cell Biol. 1976;13:247–282. [PubMed] [Google Scholar]
  20. Orias E., Flacks M., Satir B. H. Isolation and ultrastructural characterization of secretory mutants of Tetrahymena thermophila. J Cell Sci. 1983 Nov;64:49–67. doi: 10.1242/jcs.64.1.49. [DOI] [PubMed] [Google Scholar]
  21. Palen T. E., Cech T. R. Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena. Cell. 1984 Apr;36(4):933–942. doi: 10.1016/0092-8674(84)90043-6. [DOI] [PubMed] [Google Scholar]
  22. Pan W. C., Blackburn E. H. Single extrachromosomal ribosomal RNA gene copies are synthesized during amplification of the rDNA in Tetrahymena. Cell. 1981 Feb;23(2):459–466. doi: 10.1016/0092-8674(81)90141-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Pollack Y., Stein R., Razin A., Cedar H. Methylation of foreign DNA sequences in eukaryotic cells. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6463–6467. doi: 10.1073/pnas.77.11.6463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Riggs A. D. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14(1):9–25. doi: 10.1159/000130315. [DOI] [PubMed] [Google Scholar]
  26. Sneider T. W., Teague W. M., Rogachevsky L. M. S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line. Nucleic Acids Res. 1975 Oct;2(10):1685–1700. doi: 10.1093/nar/2.10.1685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stein R., Gruenbaum Y., Pollack Y., Razin A., Cedar H. Clonal inheritance of the pattern of DNA methylation in mouse cells. Proc Natl Acad Sci U S A. 1982 Jan;79(1):61–65. doi: 10.1073/pnas.79.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Turnbull J. F., Adams R. L. DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity. Nucleic Acids Res. 1976 Mar;3(3):677–695. doi: 10.1093/nar/3.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang R. Y., Huang L. H., Ehrlich M. Human placental DNA methyltransferase: DNA substrate and DNA binding specificity. Nucleic Acids Res. 1984 Apr 25;12(8):3473–3490. doi: 10.1093/nar/12.8.3473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wigler M., Levy D., Perucho M. The somatic replication of DNA methylation. Cell. 1981 Apr;24(1):33–40. doi: 10.1016/0092-8674(81)90498-0. [DOI] [PubMed] [Google Scholar]
  32. Yisraeli J., Adelstein R. S., Melloul D., Nudel U., Yaffe D., Cedar H. Muscle-specific activation of a methylated chimeric actin gene. Cell. 1986 Aug 1;46(3):409–416. doi: 10.1016/0092-8674(86)90661-6. [DOI] [PubMed] [Google Scholar]

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