Comparative analysis of DNA methylation in tobacco heterochromatic sequences (original) (raw)
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The methylation status of DNA derived from potato plants recovered from slow growth
Plant Cell, Tissue and Organ Culture, 1994
The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.
Hypomethylation and hypermethylation of the tandem repetitive 5S rRNA genes in Arabidopsis
The Plant Journal, 2008
5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.
Non-symmetrical cytosine methylation in tobacco pollen DNA
Plant Molecular Biology, 1996
We have detected sequence-specific non-symmetrical cytosine methylation within a t40 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was I x mSCpTpC, 1 x mSCpGpT, l x mSCpCpT, 2x mSCpTpT, 2x mSCpGpG, and 3x mSCpApT of which only mSCpGpG and mSCpGpT fitted the consensus sequence for symmetrical methylation in plants.
Molecular Genetics and Genomics, 2002
Bisulfite genomic sequencing was used to localise 5-methylcytosine residues (mC) in 5S rRNA genes of Arabidopsis thaliana and Secale cereale. The maps of mC distribution were compared with the previously published map of the corresponding region in Nicotiana tabacum. In all three species, the level of methylation of 5S rRNA genes was generally higher than the average for the entire genome. The ratio of 5S rDNA methylation to average overall methylation was 44%/30-33% for N. tabacum, 27%/4-6% for A. thaliana and 24%/20-22% for S. cereale. With the exception of one clone from S. cereale, no methylation-free 5S rDNA was detected. The level of methylation at different sequence motifs in 5S rDNA was calculated for N. tabacum/A. thaliana/S. cereale, and this analysis yielded the following values (expressed as a percentage of total C): mCG 90%/78%/85%, mCWG 89%/41%/ 53%, mCmCG 72%/32%/16%, mCCG 4%/2%/0%, mCHH 15%/6%/1%, where W=A or T, and H=A or C or T. Non-symmetrical methylation was almost negligible in the large genome of S. cereale but relatively frequent in N. tabacum and A. thaliana, suggesting that the strict correlation between genome size and cytosine methylation might be violated for this type of methylation. Among non-symmetrical motifs the mCWA triplets were significantly over-represented in Arabidopsis, while in tobacco this preference was not as pronounced. The differences in methylation levels in different sequence contexts might be of phylogenetic significance, but further species in related and different taxa need to be studied before firm conclusions can be drawn. Keywords Arabidopsis thaliana AE Secale cereale AE 5S rRNA gene methylation AE Bisulfite genomic sequencing AE Restriction analysis
Plant Molecular Biology, 1991
The tomato nuclear genome was determined to have a G + C content of 37 ~o which is among the lowest reported for any plant species. Non-coding regions have a G + C content even lower (32~o average) whereas coding regions are considerably richer in G + C (46~o). 5-methyl cytosine was the only modified base detected and on average 23 ~o of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20 ~/o) than mature tissues (average 25 ~o )-Mature pollen has an intermediate level ofmethylation (22 ~/o). Seeds gave the highest value (27 ~o), suggesting de novo methylation after pollination and during seed development.
European Journal of Biochemistry, 1998
We explored the possibility that the cytosine DNA methylation might be regulated by S-adenosyl-Lmethionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) pools in plant cells. In order to change the AdoHcy/AdoMet ratio (methylation index), (S)-9-(2,3-dihydroxypropyl)adenine was employed, a selective reversible inhibitor of cellular S-adenosyl-L-homocysteine hydrolase. Micromolar concentrations of the inhibitor increased dramatically (more than 1000-fold) intracellular AdoHcy levels (and concominantly the AdoHcy/AdoMet ratio) in tobacco TBY-2 cells. No toxic effect of the drug was observed and the cells displayed only marginal inhibition of growth at high AdoHcy levels. At near equal intracellular concentrations of AdoHcy and AdoMet, a significant reduction of cytosine methylation in transcribed (5SrDNA) and non-transcribed (HRS60, NTRS) sequences was observed. Interestingly, the CpCpG and CpApG trinucleotide targets appeared to be most sensitive to changes in the methylation index. Methylation of cytosine residues at CpG sites was not affected even at AdoHcy/AdoMet ratio of Ͼ 10. These results support the possible regulation of DNA methylation via AdoHcy/AdoMet metabolic pathways in plant cells.
Nucleic Acids Research, 1991
We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease Hinfl, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that Hinffl cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of Hinfl digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to Hinfl digestion. We also tested Hhall, an isoschizomer of Hinfl, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.
The Plant Journal, 2008
We have studied the inheritance of the epigenetic state of tobacco transgenes whose expression was posttranscriptionally silenced by an invertedly repeated silencer locus. We show that, in hybrids, the coding region of the target neomycin phosphotransferase (nptII) gene was almost exclusively methylated at CG configurations, and dense non-CG methylation occurred in the 3¢ untranslated region. Homologous sequences in the silencer locus were heavily methylated at both CG and non-CG motifs. After segregation of the silencer locus, the CG methylation but not the non-CG methylation of the target genes was transmitted to the progeny. In the segregants, we observed an overall increase of CG methylation in the target genes, associated with a re-distribution from the 3¢ end of the coding region towards the middle. This pattern was inherited with some fluctuation for at least two additional generations in the absence of a detectable T-DNA-derived small RNA fraction. Thus CG methylation is not cleared during meiosis and may be inherited over generations without RNA signals being present. These epi-allelic variants re-expressed the reporter gene immediately after segregation of the trigger, showing that relatively dense CG methylation (approximately 60-80%) imprinted on most of the coding region (>500 bp) did not reduce expression compared with the parental non-methylated locus. We propose that the genic CG methylation seen in euchromatic regions of the genome may originate from ancient post-transcriptional gene silencing events as a result of adventitiously produced methylationdirecting RNA molecules.