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PubMed1977

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6-7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: epsilon-N-trimethyllysine, epsilon-N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.

Amino Acids↗

PubMed1991

DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosourea.

The formation of O6-methyldeoxyguanosine (O6-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed to N-methyl-N-nitrosourea (MNU) in the drinking water at 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 mumol O6-MedGuo/mol guanine). Fundus (91 mumol/mol guanine) and pylorus (105 mumol/mol guanine) of the glandular stomach, oesophagus (124 mumol/mol guanine) and duodenum (109 mumol/mol guanine) showed lower levels of O6-MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the non-enzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione, L-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive to O6-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271-2274]. Although MNU (400 ppm) produced similar levels of O6-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methyl-purines were unequivocally identified using antibodies to O6-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosal layers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus and in the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. It is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.

Administration, Oral↗

PubMed2000

DNA stability and genomic methylation status in colonocytes isolated from methyl-donor-deficient rats.

BACKGROUND: Epidemiological studies report an inverse relationship between intake of the B vitamin folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. AIMS OF THE STUDY: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. METHODS: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionine and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. RESULTS: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocyte DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. CONCLUSIONS: Folate and methyl deficiency in vivo primarily affects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation.

Animals↗

PubMed2005

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

Cytosine↗

PubMed2006

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine.

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 microM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G1-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 microM 5-azaC may have enhanced potential for porcine NT.

Animals↗

PubMed1983

DNA sequences showing a delay in cytosine methylation after replication. Time course of methylation in synchronized mammalian cell populations and relationship to DNAase I sensitive domains.

We have shown that in several mammalian cell lines a minor fraction of cytosine methylation is delayed for up to several hours after strand synthesis and that different methylases performed the immediate and the delayed classes of DNA methylation. To investigate the time course of this delayed methylation we have used three different cell lines, two of human and one of hamster origin. These were synchronized by two different methods: mitotic detachment and double hydroxyurea blocks. A uniform picture was obtained with all three cell lines. Delayed methylation of early replicating sequences occurred while cells were still in mid-S-phase, with the maximum rate of delayed methylation occurring in cells in the second half of S and in G2. Delayed methylation seems to be complete before cells entered the next G1-phase. Limited DNAase I hydrolysis of cell nuclei was used to test whether the delay in methylation in some DNA sequences was due to high levels of transcriptional activity. However, DNA sequences exhibiting delayed methylation showed no preferential concentration in or exclusion from DNAase I hypersensitive regions.

Animals↗

PubMed2001

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.

Humans↗

PubMed1997

Reductive cleavage of the positional isomers of benzoylated and methylated methyl alpha-D-mannopyranoside.

The four O-benzoyl-tri-O-methyl positional isomers, the six di-O-benzoyl-di-O-methyl positional isomers, the four tri-O-benzoyl-O-methyl positional isomers, and the tetra-O-benzoyl derivative of methyl alpha-D-mannopyranoside were synthesized, characterized, and subjected to reductive cleavage in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate. The reactions were monitored by 1H NMR spectroscopy in order to establish the rates of the reactions and the identities of the products so formed. The tetra-O-benzoyl derivative, all tri-O-benzoyl-O-methyl positional isomers, and the 2.4-di-O-benzoyl-3.6-di-O-methyl positional isomer were fully stable to reductive-cleavage conditions, but the other di-O-benzoyl-di-O-methyl positional isomers and all O-benzoyl-tri-O-methyl positional isomers were converted to their respective 1.5-anhydro-D-mannitol derivatives via reductive cleavage of their anomeric carbon-oxygen bonds. For the mono-O-benzoyl positional isomers, the rate of reductive cleavage decreased in the order 2-O-benzoyl > 6-O-benzoyl > 3-O-benzoyl approximately 4-O-benzoyl. Moreover, all mono-O-benzoyl positional isomers were cleaved at a much faster rate than the di-O-benzoyl positional isomers. These results suggest that both anchimeric assistance and inductive effects of the ester groups contribute to the observed reaction rate. Based upon these results it is suggested that reductive cleavage of benzoylated polysaccharides might serve as a means to cleave selectively at branched residues.

Benzoates↗

PubMed1997

CpG distribution patterns in methylated and non-methylated species.

To characterize the extent of DNA methylation and its possible biological roles in a wide variety of organisms, we have analyzed gene sequences extracted from the GenBank database. Sequences of both methylated and non-methylated species were used for comparative analysis. The local CpG dinucleotide distribution near the 5' ends of genes as well as the degree of overall CpG suppression/depletion in the entire gene region were examined in all complete gene sequences for each species. We show that the distribution patterns of CpG near the 5' region of genes differ among vertebrates, invertebrates, plants and bacteria. CpG island-like peaks in CpG O/E (observed/expected ratio) were observed not only in methylated species, but also in non-methylated species. In methylated non-vertebrates, overall CpG O/E values were lower, and peaks in the CpG profile of 5' regions were larger than in non-methylated species. We discuss the implications of such biases with respect to DNA methylation.

Animals↗

PubMed1997

Enzymatic methylation of arsenic compounds: IV. In vitro and in vivo deficiency of the methylation of arsenite and monomethylarsonic acid in the guinea pig.

Using an in vitro assay which measures the transfer of a radiolabeled methyl moiety of S-[methyl-3H]adenosylmethionine ([3H]SAM) to arsenite or monomethylarsonate (MMA) to yield [methyl-3H]MMA or [methyl-3H]dimethylarsinate (DMA) respectively, guinea pig liver cytosol was found to be deficient in the enzyme activities which methylate these substrates. Moreover, when guinea pigs were given a single intraperitoneal dose of [73As]arsenate (400 micrograms/kg body weight, 25 microCi/kg body weight), very little or no methylated arsenic species were detected in the urine after cation exchange chromatography. The urine collected 0-12 h after arsenate injection contained 98% inorganic arsenic and less than 1% DMA. No MMA was detected in the 0-12 h urine. Urine collected 12-24 h after injection contained approximately 93% inorganic arsenic, 2% MMA and 3% DMA in five of the six animals studied. However, in the 12-24 h urine of one guinea pig, 17% of the radioactivity was DMA, 80% was inorganic arsenic and 3% was MMA. The guinea pig, like the marmoset and tamarin monkeys and unlike most other animals studied thus far, appears to be deficient as far as the enzyme activities that methylate inorganic arsenite. The results of these experiments suggest that there may be a genetic polymorphism associated with the enzymes that methylate inorganic arsenite.

Animals↗

PubMed2004

Methylation of the asparagine synthetase promoter in human leukemic cell lines is associated with a specific methyl binding protein.

We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.

Aspartate-Ammonia Ligase↗

PubMed1973

Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methylthymidine.

1. DNA was treated with N-methyl-N-nitrosourea at pH7-8, 37 degrees C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-(14)C-labelled N-methyl-N-nitrosourea and use of [(14)C]thymine-labelled DNA. 2. The synthesis of O(4)-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O(4)-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed.

Animals↗

PubMed2003

Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin.

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Arabidopsis↗

PubMed1980

Both the 7-methyl and the 2'-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription.

The ability of eukaryotic mRNAs to serve as primers for influenza virus RNA transcription depends on the presence of a 5'-terminal methylated can structure, the absence of which eliminates essentially all priming activity [Plotch, S. J., Bouloy, M. & Krug, R. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1618-1622]. The present study was undertaken to determine the extent to which each of the methyl groups in the cap influences the priming activity of a mRNA. To assess the importance of the 2'-O-methyl group on the penultimate base of the cap, we used several plant viral RNAs containing the monomethylated cap 0 structure, m7GpppG. Brome mosaic virus (BMV) RNA 4 stimulated influenza virus RNA transcription only about 10-15% as effectively as did globin mRNA, which has a cap with a 2'-O-methyl group. When the cap of BMV RNA 4 was enzymatically 2'-O-methylated, its priming activity was increased 14-fold. Qualitatively similar results were obtained with other plant virus RNAs. To assess the importance of the terminal 7-methyl group, BMV RNA 4 containing the cap structure GpppGm was prepared by a series of chemical and enzymatic steps. These molecules were found to be only about 15% as active in priming as BMV RNA 4 molecules containing the fully methylated cap, m7GpppGm, indicating that the terminal 7-methyl group also strongly enhances priming activity. These results indicate that the cap 1 structure (m7GpppXm) found in all mammalian cellular mRNAs is more stringently required for priming influenza virus RNA transcription than for translation in cell-free systems.

Methylation↗

PubMed1996

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Antisense Elements (Genetics)↗

PubMed1997

Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

Base Sequence↗

PubMed1988

In vitro methylation of DNA by the fumigant methyl bromide.

Investigations have shown that the physical state of the DNA strongly influences the pattern of methylation observed when DNA or a substance containing DNA is treated with the fumigant methyl bromide. 1-Methyl-adenine and 7-methylguanine were identified, after hydrolysis, as the major methylated bases of DNA which had been treated in the solid state. 3-Methylcytosine, 3-methyladenine and 7-methyladenine were found as minor products. The overall methylation pattern was similar to that observed earlier for DNA of maize and wheat which had been fumigated. In contrast, when buffer solutions of DNA were treated with methyl bromide, the N-7 position of guanine was the major site and the N-3 position of adenine was the second most important site of methylation. This result corresponds to that previously observed in similar studies with buffer solutions of DNA and other methylating agents.

Animals↗

PubMed1990

DNA methylation in eukaryotes: kinetics of demethylation and de novo methylation during the life cycle.

We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time.

Animals↗