Cpg sequences at this locus are methylated at 90 of

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Unformatted text preview: this 180-bp repeat. Primer sequences used for bisulfite sequencing are available on request. We gel-purified amplified fragments (of 650 bp) with QIAquick Gel Extraction Kit (QIAGEN), inserted them into the pGEM-T Easy Vector System (Promega) and sequenced them. CpG sequences at this locus are methylated at 90% of the sites (162 of 180) in wild-type DNA but only 1.3% in homozygous met1-3 mutants (2 of 143 sites). Considering the 3.6% deficiency in the bisulfite reaction (16 of 443 sites) deduced from reconstruction experiments, CpG methylation at these repeats is probably completely erased in homozygous met1 strains. CpNpG methylation (N = A, T or C) in wild-type was 45.6% (104 of 228 sites) and in homozygous mutants was 26.3% (59 of 190 sites). Asymmetric CpNpN in wild-type and homozygous met1 was 14.1% (190 of 1349 sites) and 10.3% (110 of 1065 sites), respectively, showing a less marked effect of the met1 mutation on both CpNpG and asymmetric sites. URLs. A. thaliana genomic sequence information is available from The Arabidopsis Information Resource website (http://www.arabidopsis.org/). A second insertion in the MET1 gene was identified in and supplied by the Syngenta Arabidopsis Insertion Library (at the Torrey Mesa Research Institute; http://www.tmri.org/). Accession numbers. We obtained MET1 sequence and A. thaliana BAC clone F9M8 sequence information from GenBank (accession numbers L10692 and AC083859, respectively). Note: Supplementary information is available on the Nature Genetics website. Acknowledgments We thank H. Vaucheret for the A. thaliana line 6b5, O. Fritsch and B. Hohn for the mutant collection, J. Finnegan for the AsMET1 line, E.J. Richards for the 180-bp repeat probe, K. Afsar and S. Lienhard for technical assistance and S. Adams, B. Hohn, U. Grossniklaus, P. King and C. Spillane for comments on the manuscript. This work was supported by grants from the Swiss Federal Office for Education and Science and the European Union to O.M.S. Competing interests statement The authors declare that they have no competing financial interests. nature genetics • volume 34 • may 2003 letter Received 13 January; accepted 7 March 2003. 1. 2. 3. 4. © 2003 Nature Publishing Group http://www.nature.com/naturegenetics 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Li, E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662–673 (2002). Kakutani, T., Munakata, K., Richards, E.J. & Hirochika, H. Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana. Genetics 151, 831–838 (1999). Jackson, J.P., Lindroth, A.M., Cao, X. & Jacobsen, S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556–560 (2002). Cao, X. & Jacobsen, S.E. Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr. Biol. 12, 1138–1144 (2002). Bartee, L., Malagnac, F. &...
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