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Retrotransposition of a Bacterial Group II Intron

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Retrotransposition of a Bacterial Group II Intron letters to nature Table 1 Appearance of hammerhead clones during selection Received 25 May; accepted 7 August 2001. 1. Pabon-Pena, L. M., Zhang, Y. & Epstein, L. M. Newt satellite 2 transcripts self-cleave by using an Round Frequency* Activity² (min-1) No.³ Progenitors§ ............................................................................................................................................................................. extended hammerhead structure. Mol. Cell. Biol. 11, 6109±6115 (1991). 2. Zhang, Y. & Epstein, L. M. Cloning and characterization of extended hammerheads from a diverse set 5 0.02 0.0026 6 0.000057 40 1/39 of caudate amphibians. Gene 172, 183±190 (1996). 7 0.17 0.026 6 0.0021 29 20/5 3. Ferbeyre, G., Smith, J. M. & Cedergren, R. Schistosome satellite DNA encodes active hammerhead 9 0.67 0.086 6 0.0022 33 23/8 11 1.00 0.166 6 0.0053 30 17/0 ribozymes. Mol. Cell. Biol. 18, 3880±3888 (1998). 12 0.98 0.322 6 0.014 55 22/1 4. Rojas, A. A. et al. Hammerhead-mediated processing of satellite pDo500 family transcripts from 13 ± 0.655 6 0.022 ± ± Dolichopoda cave crickets. Nucleic Acids Res. 28, 4037±4043 (2000). 0.0381 6 0.0031k 5. Diener, T. O. Circular RNAs: relics of precellular evolution? Proc. Natl Acad. Sci. USA 86, 9370±9374 14 0.93 0.835 6 0.054 30 19/1 (1989). 0.050 6 0.00098k 6. Williams, K. P., Ciafre, S. & Tocchini-Valentini, G. P. Selection of novel Mg2+-dependent self-cleaving 16 0.77 0.289 6 0.031 30 7/2 ribozymes. EMBO J. 14, 4551±4557 (1995). 0.0649 6 0.0069k 7. Tang, J. & Breaker, R. R. Structural diversity of self-cleaving ribozymes. Proc. Natl Acad. Sci. USA 97, ............................................................................................................................................................................. 5784±5789 (2000). * Observed frequency of hammerhead-containing clones. ² Estimated pool activity (mean 6 s.d.). 8. Thomson, J. B., Tuschl, T. & Eckstein, F. in Catalytic RNA (eds Eckstein, F. & Lilley, D. M. J.) 173±196 ³ Number of sequenced clones. (Springer, Heidelberg, 1997). § Number of distinct, independent sequences with/without hammerhead motif. 9. Ruffner, D. E., Stormo, G. D. & Uhlenbeck, O. C. Sequence requirements of the hammerhead RNA k Activity in 0.5 mM MgCl2 at pH 7.2. self-cleavage reaction. Biochemistry 29, 10695±10702 (1990). 10. Conaty, J., Hendry, P. & Lockett, T. Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration. Nucleic Acids Res. 27, 2400±2407 (1999). 11. Hampel, N. in Progress in Nucleic Acids Research and Molecular Biology (ed. Moldave, K.) 1±38 (Academic, London, 1998). 12. Been, M. D. Cis-andtrans-acting ribozymes from a human pathogen, hepatitis delta virus. Trends ef®ciently by the RT±PCR procedure we used, or must be restricted Biochem. Sci. 19, 251±256 (1994). to speci®c substrate sequences not included in our selection. Our 13. Thill, G., Vasseur, M. & Tanner, N. K. Structural and sequence elements required for the self-cleavage results are consistent with those of Tang and Breaker7, who observed activity of the hepatitis delta virus ribozyme. Biochemistry 32, 4254±4262 (1993). -1 14. Beattie, T. L., Olive, J. E. & Collins, R. A. A secondary-structure model for the self-cleaving region of many distinct ribozymes with activities lower than 0.1 min . Neurospora VS RNA. Proc. Natl Acad. Sci. USA 92, 4686±4690 (1995). 10 In contrast, another selection experiment , in which the random- 15. Gould, S. J. Wonderful Life: The Burgess Shale and the Nature of History 292±323 (Norton, New York, sequence region contained only 18 nucleotides, yielded only ham- 1989). merhead ribozymes at both low and high activities. However, the 16. Long, D. M. & Uhlenbeck, O. C. Self-cleaving catalytic RNA. FASEB J. 7, 25±30 (1993). stringent size constraint on the random-sequence region, and the constraints placed on cleavage site recognition, may have ruled out Acknowledgements the appearance of other ribozymes. There are several natural classes We thank P. Svec for assistance in cloning and sequencing, and members of the laboratory for their comments on the manuscript. This work was supported in part by a grant from of self-cleaving ribozymes other than the hammerhead, including the National Institutes of Health. J.W.S. is an investigator of the Howard Hughes Medical 11 12,13 the hairpin ribozyme , the hepatitis delta ribozyme (HDV) , and Institute. the Neurospora VS motif14. We wondered why none of these Correspondence and requests for materials should be addressed to J.W.S. alternative highly active ribozymes was observed in our selection. (e-mail: [email protected]). These ribozymes are all signi®cantly more complex than the hammerhead ribozyme, and are thus expected to occur much less frequently in selection experiments, in the absence of selective factors other than rate of cleavage. Thus, if selective pressure requires only that self-cleavage activity be in the range of 0.1± 1.0 min-1, the expected result, both in vitro and in nature, is the emergence of the hammerhead ribozyme as the most probable ................................................................. solution. Our results show that, despite the dominance of con- tingency (historical accident) in some recent discussions of evolu- corrections tionary mechanisms15, purely chemical constraints (that is, the ability of only certain sequences to carry out particular functions) can lead to the repeated evolution of the same macromolecular structures. M Retrotransposition of a bacterial Methods group II intron Selection procedure Benoit Cousineau, Stacey Lawrence, Dorie Smith & Marlene Belfort Pool RNA dissolved in 10 mM Tris-HCl buffer at pH 8.0 and 1 mM EDTA (TE) was heated to 85 8C then cooled to ambient temperature. Cleavage reactions were started by adding Nature 404, 1018±1021 (2000). pool RNA to reaction buffer such that the ®nal composition was 1 mM RNA, 5 mM MgCl2, .................................................................................................................................. 50 mM Tris-HCl at pH 7.8, 10 mM DTTand 1 U ml-1 RNasin. Reaction times were 17±20 h In this Letter, we demonstrated active group II intron transposition at 26 8C in rounds 1±5 and in the subsequent rounds were adjusted to produce 1% to multiple ectopic sites and showed that movement to these cleavage. Reactions were stopped by addition of two volumes of 8 M urea, 2 mM Tris-HCl at pH 7.5 and 75 mM EDTA. Samples were resolved by electrophoresis in 8% denaturing new sites occurs via an RNA intermediate in a process termed polyacrylamide gels alongside a marker RNA to identify the position of the cleaved retrotransposition. Of interest was whether retrotransposition products; this region was excised and the RNAwas isolated. RNAs were reverse transcribed occurs by reverse splicing of the intron RNA into RNA, single- using SuperScript II (Gibco-BRL), ampli®ed by PCR and transcribed with T7 RNA stranded DNA and/or double-stranded DNA targets. Our results polymerase (0.5 mM nucleoside triphosphates, 40 mM Tris-HCl at pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol and 1 U ml-1 RNasin) in the presence of 50 mMof suggested that retrotransposition occurs predominantly by the the blocking oligonucleotide (59-TACTGACTGGGTCCAG-39). After transcription, RNAs intron reverse-splicing into an RNA target. However, subsequent were puri®ed by denaturing polyacrylamide gels, eluted, ethanol precipitated, dissolved in experiments using a different intron donor have indicated that TE, and stored at -80 8C. the selection method for retrotransposition events can strongly bias the interpretation of pathway (K. Ichiyanagi, A. Beauregard and Cleavage site determination M.B., unpublished results). The new data do not point to RNA- RNAs were denatured with heat, cooled to ambient temperature, incubated in targeting as the principal means for intron retrotransposition, and cleavage buffer, and then reverse transcribed with a 59-32P-labelled primer (59- CGTTGTGGTCTATC-39). Samples mixed with formamide stop buffer were analysed DNA-based pathways must be considered as playing an important by electrophoresis in 12% denaturing polyacrylamide gels. role. M 84 © 2001 Macmillan Magazines Ltd NATURE | VOL 414 | 1 NOVEMBER 2001 | www.nature.com letters to nature pol IV (20 nM) or pol V (25±50 nM). The running-start nucleotide was absent in polymerase activated by UmuD9, RecA, and SSB and is specialized for translesion replication. J. Biol. standing-start reactions. Reactions were run at 37 8C for 5 min and quenched by adding Chem. 274, 31763±31766 (1999). 20 ml of 20 mM EDTA in 95% formamide. Primer±template extension products were heat 24. Radman, M. Enzymes of evolutionary change. Nature 401, 866±869 (1999). denatured and run on a 16% polyacrylamide denaturing gel. We measured integrated gel- 25. McDonald, J. P. et al. Novel human and mouse homologs of Saccharomyces cerevisiae DNA band intensities by phosphorimaging using ImageQuant software (Molecular Dynamics). polymerase h. Genomics 60, 20±30 (1999). Nucleotide incorporation rates opposite both undamaged and lesion target sites were 26. Gerlach, V. L. et al. Human and mouse homologs of Escherichia coli DinB (DNA polymerase IV), members of the UmuC/DinB superfamily. Proc. Natl Acad. Sci. USA 96, 11922±11927 (1999). determined, and apparent Vmax/Km values and nucleotide misincorporation frequencies were calculated as described16. We carried out ®delity measurements using standing-start 27. Naktinis, V., Turner, J. & O'Donnell, M. A molecular switch in a replication machine de®ned by an reactions on undamaged linear M13mp7 DNA templates annealed to 30mer 32P-labelled internal competition for protein rings. Cell 84, 137±145 (1996). primers with ATP (100 mM). The following template sequences were used: 39¼GTTGC 28. Jing, Y., Kao, J. F.-L. & Taylor, J.-S. Thermodynamic and base-pairing studies of matched and CG¼;39¼CGTAGCC¼;39TCGTAGC¼;39TATCAAC, where the base in bold is the mismatched DNA dodecamer duplexes containing cis±syn, (6±4) and Dewar photoproducts of TT.
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