On the impossibility of the incorporation of 5-methylcytosine and its nucleosides into higher plant DNA

No radioactivity was detected in 5-methylcytosine isolated from wheat DNA after incubation of wheat seedlings with 3H-labelled 5-methylcytosine, 5-methylcytidine and 5-methyldeoxycytidine. No label from 3H-5-methylcytosine was found in DNA of seedlings. After incubation of seedlings with 3H-labelled nucleosides of 5-methylcytosine, radioactivity was discovered only in thymine of DNA. Thus 5-methylcytosine and its nucleosides can not be used in plants as direct precursors of 5-methyl cytosine residues in DNA, but nucleosides of 5-methylcytosine may be deaminated to thymidine (or deoxythymidine) and subsequently incorporated into DNA.     

Non-enzymatic DNA methylation by S-adenosylmethionine results in the formation of minor thymine residues and 5-methylcytosine from cytosine

It was found that nonenzymatic DNA methylation proceeds in water solution in the presence of S-adenosylmethionine (AdoMet). The main reaction products are thymine and 5-methylcytosine residues. It was shown that labelled thymine residues are formed also upon DNA incubation in the presence of [methyl-14C]methionine as well as [methyl-14C]cobalamine. Only cytosine reacts with AdoMet resulting in thymine production. AdoMet may be a potential mutagen that induces GC----AT transitions during DNA replication in the cell.     

The kinetics of DNA methylation in cultures of a mouse adrenal cell line

Direct measurements of the methylation of newly-synthesized DNA were made in cultures of a clonal mouse adrenal cortex cell line, Y129OS3, by (1) following the incorporation of radioactivity from methionine-(methyl)-C¹⁴ into a segment of DNA which had been density-labeled with bromouracil and (2) labeling DNA cytosine with C¹⁴-deoxycytidine and then following the appearance of radioactivity in DNA 5-methylcytosine. The results establish that during exponential growth the DNA of this cell line is methylated entirely within a few minutes of its synthesis. Using the second technique described above accurate, sensitive measurements of DNA methylation levels can be made by comparing radioactivity in 5-methylcytosine to radioactivity in cytosine plus 5-methylcytosine. In this cell line 5-methylcytosine accounts for 4.3 ± 0.2% of the DNA cytosine. Some apparent contradictions between these results and those of other workers are discussed.     

Mechanism of counteraction of aflatoxin toxicity by Thymine and Folic acid inBacillus megaterium

Thymine and cytosine as well as the intermediates in pyrimidine biosynthetic pathway dihydroorotic acid, orotic acid, carbamyl aspartate and 5'-uridine monophosphate and folic acid, was synergistic. 2-14C-Thymine, 6-14C-orotic acid and 14C-formate but not 2-14C-uracil, were incorporated into DNA more in the presence of aflatoxin. These findings indicated that aflatoxin inhibited the pyrimidine base synthesis which could be overcome to a great extent by the addition of thymine and folic acid to aflatoxin-treated cells.     

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos

Nuclei isolated from the developing sea urchin embryo $\text{Paracentrotus lividus}$ and incubated in the presence of [³H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine.

The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).     

Enzymic removal of 5-methylcytosine from poly(dG-5-methyl-dC) by HeLa cell nuclear extracts is not by a DNA glycosylase.

A recent report in this journal [Vairapandi, M. and Duker, N.J. (1993) Nucleic Acids Res. 21, 5323-5327) presented evidence of an activity in HeLa cell nuclear extracts that released radiolabeled material from a poly(dG.dC) polymer that had been methylated and simultaneously labeled on cytosine residues by incubation with a CpG-specific DNA methylase and [methyl-3H]S-adenosylmethionine. Based on chromatographic evidence that the released products were thymine and 5-methylcytosine and on f1p4olabeling data suggesting a concomitant increase in abasic sites, the authors concluded that the releasing activity was a 5-methylcytosine-specific glycosylase and that the solubilized 5-methylcytosine was converted to thymine by a nuclear deaminase. We have confirmed that HeLa nuclear extracts promote release of ethanol-soluble radioactivity from a methyl-labeled poly(dG-5-methyl-dC)polymer, but the products released were neither 5-methylcytosine nor thymine. Furthermore, free 5-methylcytosine was not deaminated by incubation with the nuclear extract. The labeled compound released initially from the polymer appeared to be 5-methyl-deoxycytidine monophosphate, which was converted to 5-methyl-deoxycytidine, thymidine monophosphate, and/or thymidine by further incubation with the nuclear extract. The activity responsible for the release, therefore, was a nuclease. Release of 32P-labeled nucleotides from a 32P-labeled poly(dG-dC) polymer suggested, furthermore, that the activity was not specific for methylated DNA.     

Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.     

Selective Hypermethylation of Transcribed Nucleosomal DNA by Sodium Butyrate

Previous studies have shown that treatment of cultured fibroblasts with millimolar concentrations of sodium butyrate results in increased methylation of cytosine residues in DNA. In this study, active nucleosomes were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its constituent bases and subjected to HPLC analysis in order to determine the 5-methylcytosine content. In control cells, the active nucleosomal DNA was hypomethylated (0.97 ± 0.27% 5-methylcytosine) when compared with the inactive DNA fraction (1.61 ± 0.15%). This result was not unexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resulted in increased methylation of the active nucleosomal DNA such that it was comparable to that of the inactive fraction of control cells (1.73 ± 0.02% 5-methylcytosine). A much smaller increase in 5-methylcytosine content was detected in the inactive DNA fraction of sodium butyrate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrate followed by a chase in butyrate-free medium for up to 120 h failed to reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days.     

Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.     

Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A high-performance liquid chromatographic method to separate five major bases (cytosine, thymine, guanine, adenine, and uracil) and three minor methylated bases (5-methylcytosine, N6-methyladenine, and 7-methylguanine) has been developed using a volatile mobile phase under isocratic conditions. It is extended to quantitate 5-methylcytosine in trace amounts (1 in 20,000 cytosine residues). The suitability of the method has been verified by estimating 5-methylcytosine in DNAs of phi X174 and pBR322. The method has been applied to quantitate the extent of cytosine methylation in DNA of larval silk glands of Bombyx mori. Our results confirm that the pupal DNA of Drosophila melanogaster does not contain detectable amounts of 5-methylcytosine.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

Deoxyribonucleic acid methylation and development in Caulobacter bacteroides

The stalked bacterium Caulobacter bacteroides, which displays a series of developmental changes during its life cycle, was shown to contain the methylated deoxyribonucleic acid (DNA) nucleotide bases N(6)-methyladenine and 5-methylcytosine as well as the enzymes required for the synthesis of these bases. A difference in the specific activity of these enzymes was observed between swarmer cells, which are low in activity, and stalked cells, which are high in activity. This difference was not reflected in the methylation patterns in the DNA of the two cell types since C. bacteroides DNA is essentially completely methylated with respect to C. bacteroides methylases.     

Hypomethylation of repair patches in HeLa cells

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.     

A Study of the Effects of Hydrostatic Pressure on Macromolecular Synthesis in Escherichia coli

In cultures of Escherichia coli 15 (thymine^-, leucine^-) which were incubated at high hydrostatic pressures, cell division occurred only at pressures below 430 atm but in a somewhat synchronous fashion at around 250 atm. The rate of leucine-¹⁴C incorporation into a macromolecular fraction of the cells diminished to a zero value at about 580 atm and that of uracil-¹⁴C incorporation to a zero value at about 770 atm. The rate of thymine-¹⁴C incorporation at pressures around 330 atm was that to be expected with a culture in which DNA synthesis is somewhat synchronous. At pressures above 500 atm, thymine-¹⁴C was incorporated only over the initial part of the pressure incubation and further incorporation under pressure was not observed no matter how long the duration of the incubation. We present evidence along several lines that the thymine incorporation kinetics reflect an effect of pressure on a locus at the origin (or termination) of a replication of the bacterial chromosome. The recovery of cell division and of the incorporation rates upon release of pressure were found to depend on the magnitude of the pressure and the duration of the pressure incubation.     

Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries

After emergence female houseflies were fed for 4 days on a diet containing ¹⁴C-orotic acid and ³H-thymidine-5-triphosphate, or ³H-leucine. Nucleic acids and ribosomes were then isolated from the ovaries and studied by MAK column chromatography and sedimentation analysis respectively. The ultraviolet absorption and radioactivity of the fractions were also measured. After MAK column chromatography, the u.v. elution pattern showed that only tow distinct peaks, corresponding to tRNA and rRNA were present. A similar elution pattern was obtained by measuring the ¹⁴C from ¹⁴C-orotic acid incorporated into the RNA. Because of the small quantity present, DNA was not measurable by u.v. absorption, but by determining the incorporation of ³H from ³H-TTP, its presence was clearly evident.Sedimentation analysis of ovarian ribosomes revealed four polymeric forms besides subunits and monomers. The incorporation of ¹⁴C-orotic acid and ³H-leucine into the ribosomes was used to follow the synthesis of rRNA and rProtein respectively. Sucrose density gradient centrifugation of the rRNA indicated that the ovarian rRNA consisted primarily of 28 and 18 S particles.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.     

Intragenome distribution of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants (Gossypium hirsutum L.)

Fractionation of DNA of healthy and wilt-infected cotton plants has been carried out according to the reassociation kinetics and the content of GC and 5-methylcytosine in the resulting fractions has been studied. The genome of cotton plant was found to be methylated quite unevenly. The GC rich (GC=64.7 mole%) fraction of highly reiterated sequences (C ₀ t=0–3.7×10^-2) has a high content of 5-methylcytosine (5.8 mole%), whereas the methylation degree of the fraction of unique sequences (C ₀ t487) is very low (the 5-methylcytosine content is about 0.5 mole%). In plants being infected with wilt, the 5-methylcytosine content in DNA of cotton leaves decreases two-fold; no other changes in the structure and molecular population of DNA has been found. The sharp change in the 5-methylcytosine content in DNA of infected plants takes place at the expense of the decrease in the 5-methylcytosine content in fractions of highly reiterated sequences. The methylation degree of unique sequences (structural genes) remains unchanged.