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. 1998 Sep 15;26(18):4230–4240. doi: 10.1093/nar/26.18.4230

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

L A Klobutcher 1, S E Gygax 1, J D Podoloff 1, J R Vermeesch 1, C M Price 1, C M Tebeau 1, C L Jahn 1
PMCID: PMC147820  PMID: 9722644

Abstract

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

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Selected References

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  1. Baird S. E., Fino G. M., Tausta S. L., Klobutcher L. A. Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development. Mol Cell Biol. 1989 Sep;9(9):3793–3807. doi: 10.1128/mcb.9.9.3793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baird S. E., Klobutcher L. A. Characterization of chromosome fragmentation in two protozoans and identification of a candidate fragmentation sequence in Euplotes crassus. Genes Dev. 1989 May;3(5):585–597. doi: 10.1101/gad.3.5.585. [DOI] [PubMed] [Google Scholar]
  3. Bednenko J., Melek M., Greene E. C., Shippen D. E. Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation. EMBO J. 1997 May 1;16(9):2507–2518. doi: 10.1093/emboj/16.9.2507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boswell R. E., Klobutcher L. A., Prescott D. M. Inverted terminal repeats are added to genes during macronuclear development in Oxytricha nova. Proc Natl Acad Sci U S A. 1982 May;79(10):3255–3259. doi: 10.1073/pnas.79.10.3255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Coyne R. S., Yao M. C. Evolutionary conservation of sequences directing chromosome breakage and rDNA palindrome formation in tetrahymenine ciliates. Genetics. 1996 Dec;144(4):1479–1487. doi: 10.1093/genetics/144.4.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fan X., Price C. M. Coordinate regulation of G- and C strand length during new telomere synthesis. Mol Biol Cell. 1997 Nov;8(11):2145–2155. doi: 10.1091/mbc.8.11.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Florian V., Klein A. A nascent micronuclear pseudogene in the ciliate Euplotes crassus. Nucleic Acids Res. 1996 Aug 15;24(16):3195–3200. doi: 10.1093/nar/24.16.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frels J. S., Jahn C. L. DNA rearrangements in Euplotes crassus coincide with discrete periods of DNA replication during the polytene chromosome stage of macronuclear development. Mol Cell Biol. 1995 Dec;15(12):6488–6495. doi: 10.1128/mcb.15.12.6488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frels J. S., Tebeau C. M., Doktor S. Z., Jahn C. L. Differential replication and DNA elimination in the polytene chromosomes of Euplotes crassus. Mol Biol Cell. 1996 May;7(5):755–768. doi: 10.1091/mbc.7.5.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ghosh S., Jaraczewski J. W., Klobutcher L. A., Jahn C. L. Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes. Nucleic Acids Res. 1994 Jan 25;22(2):214–221. doi: 10.1093/nar/22.2.214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs M. E., Klobutcher L. A. The long and the short of developmental DNA deletion in Euplotes crassus. J Eukaryot Microbiol. 1996 Nov-Dec;43(6):442–452. doi: 10.1111/j.1550-7408.1996.tb04503.x. [DOI] [PubMed] [Google Scholar]
  14. Jahn C. L., Erbeznik M., Jaraczewski J. W., Melek M., Shippen D. E. Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity. Gene. 1994 Dec 30;151(1-2):231–235. doi: 10.1016/0378-1119(94)90662-9. [DOI] [PubMed] [Google Scholar]
  15. Jahn C. L., Ling Z., Tebeau C. M., Klobutcher L. A. An unusual histone H3 specific for early macronuclear development in Euplotes crassus. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1332–1337. doi: 10.1073/pnas.94.4.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jahn C. L., Nilles L. A., Krikau M. F. Organization of the Euplotes crassus micronuclear genome. J Protozool. 1988 Nov;35(4):590–601. doi: 10.1111/j.1550-7408.1988.tb04157.x. [DOI] [PubMed] [Google Scholar]
  17. Klobutcher L. A. Developmentally excised DNA sequences in Euplotes crassus capable of forming G quartets. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1979–1983. doi: 10.1073/pnas.92.6.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Klobutcher L. A., Herrick G. Developmental genome reorganization in ciliated protozoa: the transposon link. Prog Nucleic Acid Res Mol Biol. 1997;56:1–62. doi: 10.1016/s0079-6603(08)61001-6. [DOI] [PubMed] [Google Scholar]
  19. Klobutcher L. A., Huff M. E., Gonye G. E. Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova. Nucleic Acids Res. 1988 Jan 11;16(1):251–264. doi: 10.1093/nar/16.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klobutcher L. A., Swanton M. T., Donini P., Prescott D. M. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A. 1981 May;78(5):3015–3019. doi: 10.1073/pnas.78.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lingner J., Cech T. R. Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10712–10717. doi: 10.1073/pnas.93.20.10712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Madireddi M. T., Coyne R. S., Smothers J. F., Mickey K. M., Yao M. C., Allis C. D. Pdd1p, a novel chromodomain-containing protein, links heterochromatin assembly and DNA elimination in Tetrahymena. Cell. 1996 Oct 4;87(1):75–84. doi: 10.1016/s0092-8674(00)81324-0. [DOI] [PubMed] [Google Scholar]
  23. Melek M., Greene E. C., Shippen D. E. Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage. Mol Cell Biol. 1996 Jul;16(7):3437–3445. doi: 10.1128/mcb.16.7.3437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ochman H., Gerber A. S., Hartl D. L. Genetic applications of an inverse polymerase chain reaction. Genetics. 1988 Nov;120(3):621–623. doi: 10.1093/genetics/120.3.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Prescott D. M., DuBois M. L. Internal eliminated segments (IESs) of Oxytrichidae. J Eukaryot Microbiol. 1996 Nov-Dec;43(6):432–441. doi: 10.1111/j.1550-7408.1996.tb04502.x. [DOI] [PubMed] [Google Scholar]
  26. Prescott D. M. The DNA of ciliated protozoa. Microbiol Rev. 1994 Jun;58(2):233–267. doi: 10.1128/mr.58.2.233-267.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Price C. M. Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein. Mol Cell Biol. 1990 Jul;10(7):3421–3431. doi: 10.1128/mcb.10.7.3421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Qian L., Wilkinson M. DNA fragment purification: removal of agarose 10 minutes after electrophoresis. Biotechniques. 1991 Jun;10(6):736–738. [PubMed] [Google Scholar]
  29. Roth M., Lin M., Prescott D. M. Large scale synchronous mating and the study of macronuclear development in Euplotes crassus. J Cell Biol. 1985 Jul;101(1):79–84. doi: 10.1083/jcb.101.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roth M., Prescott D. M. DNA intermediates and telomere addition during genome reorganization in Euplotes crassus. Cell. 1985 Jun;41(2):411–417. doi: 10.1016/s0092-8674(85)80014-3. [DOI] [PubMed] [Google Scholar]
  31. Seegmiller A., Williams K. R., Hammersmith R. L., Doak T. G., Witherspoon D., Messick T., Storjohann L. L., Herrick G. Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins. Mol Biol Evol. 1996 Dec;13(10):1351–1362. doi: 10.1093/oxfordjournals.molbev.a025581. [DOI] [PubMed] [Google Scholar]
  32. Shippen-Lentz D., Blackburn E. H. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. doi: 10.1126/science.1689074. [DOI] [PubMed] [Google Scholar]
  33. Shippen-Lentz D., Blackburn E. H. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. doi: 10.1126/science.1689074. [DOI] [PubMed] [Google Scholar]
  34. Tausta S. L., Klobutcher L. A. Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus. Nucleic Acids Res. 1990 Feb 25;18(4):845–853. doi: 10.1093/nar/18.4.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vermeesch J. R., Price C. M. Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus. Mol Cell Biol. 1994 Jan;14(1):554–566. doi: 10.1128/mcb.14.1.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vermeesch J. R., Williams D., Price C. M. Telomere processing in Euplotes. Nucleic Acids Res. 1993 Nov 25;21(23):5366–5371. doi: 10.1093/nar/21.23.5366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Yu G. L., Bradley J. D., Attardi L. D., Blackburn E. H. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature. 1990 Mar 8;344(6262):126–132. doi: 10.1038/344126a0. [DOI] [PubMed] [Google Scholar]

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