Abstract
We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.
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- Allen N. D., Norris M. L., Surani M. A. Epigenetic control of transgene expression and imprinting by genotype-specific modifiers. Cell. 1990 Jun 1;61(5):853–861. doi: 10.1016/0092-8674(90)90195-k. [DOI] [PubMed] [Google Scholar]
- Bestor T. H., Ingram V. M. Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5559–5563. doi: 10.1073/pnas.80.18.5559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bestor T., Laudano A., Mattaliano R., Ingram V. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J Mol Biol. 1988 Oct 20;203(4):971–983. doi: 10.1016/0022-2836(88)90122-2. [DOI] [PubMed] [Google Scholar]
- Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
- Boyes J., Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell. 1991 Mar 22;64(6):1123–1134. doi: 10.1016/0092-8674(91)90267-3. [DOI] [PubMed] [Google Scholar]
- Carlson L. L., Page A. W., Bestor T. H. Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting. Genes Dev. 1992 Dec;6(12B):2536–2541. doi: 10.1101/gad.6.12b.2536. [DOI] [PubMed] [Google Scholar]
- Cedar H. DNA methylation and gene activity. Cell. 1988 Apr 8;53(1):3–4. doi: 10.1016/0092-8674(88)90479-5. [DOI] [PubMed] [Google Scholar]
- Cedar H., Solage A., Glaser G., Razin A. Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI. Nucleic Acids Res. 1979;6(6):2125–2132. doi: 10.1093/nar/6.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engler P., Haasch D., Pinkert C. A., Doglio L., Glymour M., Brinster R., Storb U. A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci. Cell. 1991 Jun 14;65(6):939–947. doi: 10.1016/0092-8674(91)90546-b. [DOI] [PubMed] [Google Scholar]
- Finnegan E. J., Dennis E. S. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana. Nucleic Acids Res. 1993 May 25;21(10):2383–2388. doi: 10.1093/nar/21.10.2383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foss H. M., Roberts C. J., Claeys K. M., Selker E. U. Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation. Science. 1993 Dec 10;262(5140):1737–1741. doi: 10.1126/science.7505062. [DOI] [PubMed] [Google Scholar]
- Giordano M., Mattachini M. E., Cella R., Pedrali-Noy G. Purification and properties of a novel DNA methyltransferase from cultured rice cells. Biochem Biophys Res Commun. 1991 Jun 14;177(2):711–719. doi: 10.1016/0006-291x(91)91846-5. [DOI] [PubMed] [Google Scholar]
- 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]
- Gruenbaum Y., Naveh-Many T., Cedar H., Razin A. Sequence specificity of methylation in higher plant DNA. Nature. 1981 Aug 27;292(5826):860–862. doi: 10.1038/292860a0. [DOI] [PubMed] [Google Scholar]
- Hattman S., Schlagman S., Cousens L. Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in partial protection of bacteriophage lambda against restriction by cells containing the N-3 drug-resistance factor. J Bacteriol. 1973 Sep;115(3):1103–1107. doi: 10.1128/jb.115.3.1103-1107.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoffman J. L. Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine. Biochemistry. 1986 Jul 29;25(15):4444–4449. doi: 10.1021/bi00363a041. [DOI] [PubMed] [Google Scholar]
- Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
- Houlston C. E., Cummings M., Lindsay H., Pradhan S., Adams R. L. DNA substrate specificity of pea DNA methylase. Biochem J. 1993 Aug 1;293(Pt 3):617–624. doi: 10.1042/bj2930617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leonhardt H., Page A. W., Weier H. U., Bestor T. H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell. 1992 Nov 27;71(5):865–873. doi: 10.1016/0092-8674(92)90561-p. [DOI] [PubMed] [Google Scholar]
- Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
- Marinus M. G., Morris N. R. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 1973 Jun;114(3):1143–1150. doi: 10.1128/jb.114.3.1143-1150.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Messeguer R., Ganal M. W., Steffens J. C., Tanksley S. D. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA. Plant Mol Biol. 1991 May;16(5):753–770. doi: 10.1007/BF00015069. [DOI] [PubMed] [Google Scholar]
- Peters R. Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim Biophys Acta. 1986 Dec 22;864(3-4):305–359. doi: 10.1016/0304-4157(86)90003-1. [DOI] [PubMed] [Google Scholar]
- Pruitt R. E., Meyerowitz E. M. Characterization of the genome of Arabidopsis thaliana. J Mol Biol. 1986 Jan 20;187(2):169–183. doi: 10.1016/0022-2836(86)90226-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Sapienza C., Paquette J., Tran T. H., Peterson A. Epigenetic and genetic factors affect transgene methylation imprinting. Development. 1989 Sep;107(1):165–168. doi: 10.1242/dev.107.1.165. [DOI] [PubMed] [Google Scholar]
- Theiss G., Schleicher R., Schimpff-Weiland G., Follmann H. DNA methylation in wheat. Purification and properties of DNA methyltransferase. Eur J Biochem. 1987 Aug 17;167(1):89–96. doi: 10.1111/j.1432-1033.1987.tb13307.x. [DOI] [PubMed] [Google Scholar]
- Vongs A., Kakutani T., Martienssen R. A., Richards E. J. Arabidopsis thaliana DNA methylation mutants. Science. 1993 Jun 25;260(5116):1926–1928. doi: 10.1126/science.8316832. [DOI] [PubMed] [Google Scholar]
- Yesufu H. M., Hanley A., Rinaldi A., Adams R. L. DNA methylase from Pisum sativum. Biochem J. 1991 Jan 15;273(Pt 2):469–475. doi: 10.1042/bj2730469. [DOI] [PMC free article] [PubMed] [Google Scholar]