Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Dec;17(12):7237–7247. doi: 10.1128/mcb.17.12.7237

Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila.

P Blomberg 1, C Randolph 1, C H Yao 1, M C Yao 1
PMCID: PMC232581  PMID: 9372956

Abstract

We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (approximately 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription. It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

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. Benbow R. M., Zhao J., Larson D. D. On the nature of origins of DNA replication in eukaryotes. Bioessays. 1992 Oct;14(10):661–670. doi: 10.1002/bies.950141004. [DOI] [PubMed] [Google Scholar]
  3. Broach J. R., Li Y. Y., Feldman J., Jayaram M., Abraham J., Nasmyth K. A., Hicks J. B. Localization and sequence analysis of yeast origins of DNA replication. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1165–1173. doi: 10.1101/sqb.1983.047.01.132. [DOI] [PubMed] [Google Scholar]
  4. Bruns P. J., Katzen A. L., Martin L., Blackburn E. H. A drug-resistant mutation in the ribosomal DNA of Tetrahymena. Proc Natl Acad Sci U S A. 1985 May;82(9):2844–2846. doi: 10.1073/pnas.82.9.2844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Celniker S. E., Sweder K., Srienc F., Bailey J. E., Campbell J. L. Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2455–2466. doi: 10.1128/mcb.4.11.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Challoner P. B., Amin A. A., Pearlman R. E., Blackburn E. H. Conserved arrangements of repeated DNA sequences in nontranscribed spacers of ciliate ribosomal RNA genes: evidence for molecular coevolution. Nucleic Acids Res. 1985 Apr 11;13(7):2661–2680. doi: 10.1093/nar/13.7.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clyne R. K., Kelly T. J. Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe. EMBO J. 1995 Dec 15;14(24):6348–6357. doi: 10.1002/j.1460-2075.1995.tb00326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coyne R. S., Chalker D. L., Yao M. C. Genome downsizing during ciliate development: nuclear division of labor through chromosome restructuring. Annu Rev Genet. 1996;30:557–578. doi: 10.1146/annurev.genet.30.1.557. [DOI] [PubMed] [Google Scholar]
  10. DePamphili M. L. How transcription factors regulate origins of DNA replication in eukaryotic cells. Trends Cell Biol. 1993 May;3(5):161–167. doi: 10.1016/0962-8924(93)90137-p. [DOI] [PubMed] [Google Scholar]
  11. DePamphilis M. L. Eukaryotic DNA replication: anatomy of an origin. Annu Rev Biochem. 1993;62:29–63. doi: 10.1146/annurev.bi.62.070193.000333. [DOI] [PubMed] [Google Scholar]
  12. Du C., Sanzgiri R. P., Shaiu W. L., Choi J. K., Hou Z., Benbow R. M., Dobbs D. L. Modular structural elements in the replication origin region of Tetrahymena rDNA. Nucleic Acids Res. 1995 May 25;23(10):1766–1774. doi: 10.1093/nar/23.10.1766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Engberg J., Din N., Saiga H., Higashinakagawa T. Nucleotide sequence of the 5'-terminal coding region for pre-rRNA and mature 17S rRNA in Tetrahymena thermophila rDNA. Nucleic Acids Res. 1984 Jan 25;12(2):959–972. doi: 10.1093/nar/12.2.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Engberg J., Nielsen H. Complete sequence of the extrachromosomal rDNA molecule from the ciliate Tetrahymena thermophila strain B1868VII. Nucleic Acids Res. 1990 Dec 11;18(23):6915–6919. doi: 10.1093/nar/18.23.6915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engberg J. The ribosomal RNA genes of Tetrahymena: structure and function. Eur J Cell Biol. 1985 Jan;36(1):133–151. [PubMed] [Google Scholar]
  16. Gaertig J., Gorovsky M. A. Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9196–9200. doi: 10.1073/pnas.89.19.9196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kahn R. W., Andersen B. H., Brunk C. F. Transformation of Tetrahymena thermophila by microinjection of a foreign gene. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9295–9299. doi: 10.1073/pnas.90.20.9295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kapler G. M., Blackburn E. H. A weak germ-line excision mutation blocks developmentally controlled amplification of the rDNA minichromosome of Tetrahymena thermophila. Genes Dev. 1994 Jan;8(1):84–95. doi: 10.1101/gad.8.1.84. [DOI] [PubMed] [Google Scholar]
  20. Kapler G. M. Developmentally regulated processing and replication of the Tetrahymena rDNA minichromosome. Curr Opin Genet Dev. 1993 Oct;3(5):730–735. doi: 10.1016/s0959-437x(05)80091-7. [DOI] [PubMed] [Google Scholar]
  21. Kapler G. M., Orias E., Blackburn E. H. Tetrahymena thermophila mutants defective in the developmentally programmed maturation and maintenance of the rDNA minichromosome. Genetics. 1994 Jun;137(2):455–466. doi: 10.1093/genetics/137.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. King B. O., Yao M. C. Tandemly repeated hexanucleotide at Tetrahymena rDNA free end is generated from a single copy during development. Cell. 1982 Nov;31(1):177–182. doi: 10.1016/0092-8674(82)90417-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Løvlie A., Haller B. L., Orias E. Molecular evidence for somatic recombination in the ribosomal DNA of Tetrahymena thermophila. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5156–5160. doi: 10.1073/pnas.85.14.5156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  27. Miyahara K., Hashimoto N., Higashinakagawa T., Pearlman R. E. Common sequence elements are important for transcription and replication of the extrachromosomal rRNA-encoding genes of Tetrahymena. Gene. 1993 May 30;127(2):209–213. doi: 10.1016/0378-1119(93)90721-e. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Palen T. E., Cech T. R. Transcribed and non-transcribed regions of Tetrahymena ribosomal gene chromatin have different accessibilities to micrococcal nuclease. Nucleic Acids Res. 1983 Apr 11;11(7):2077–2091. doi: 10.1093/nar/11.7.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Pan W. J., Gallagher R. C., Blackburn E. H. Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter. Mol Cell Biol. 1995 Jun;15(6):3372–3381. doi: 10.1128/mcb.15.6.3372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spangler E. A., Blackburn E. H. The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin. J Biol Chem. 1985 May 25;260(10):6334–6340. [PubMed] [Google Scholar]
  33. Sweeney R., Chen L., Yao M. C. An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence. Mol Cell Biol. 1994 Jun;14(6):4203–4215. doi: 10.1128/mcb.14.6.4203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sweeney R., Yao C. H., Yao M. C. A mutation in the large subunit ribosomal RNA gene of Tetrahymena confers anisomycin resistance and cold sensitivity. Genetics. 1991 Feb;127(2):327–334. doi: 10.1093/genetics/127.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Yaeger P. C., Orias E., Shaiu W. L., Larson D. D., Blackburn E. H. The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila. Mol Cell Biol. 1989 Feb;9(2):452–460. doi: 10.1128/mcb.9.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yao M. C., Kimmel A. R., Gorovsky M. A. A small number of cistrons for ribosomal RNA in the germinal nucleus of a eukaryote, Tetrahymena pyriformis. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3082–3086. doi: 10.1073/pnas.71.8.3082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yao M. C. Programmed DNA deletions in Tetrahymena: mechanisms and implications. Trends Genet. 1996 Jan;12(1):26–30. doi: 10.1016/0168-9525(96)81385-0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Yao M. C., Yao C. H. Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9493–9497. doi: 10.1073/pnas.88.21.9493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yao M. C., Zheng K., Yao C. H. A conserved nucleotide sequence at the sites of developmentally regulated chromosomal breakage in Tetrahymena. Cell. 1987 Mar 13;48(5):779–788. doi: 10.1016/0092-8674(87)90075-4. [DOI] [PubMed] [Google Scholar]
  44. Yu G. L., Hasson M., Blackburn E. H. Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5151–5155. doi: 10.1073/pnas.85.14.5151. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES