Methylation Pattern of Lambda Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.     

Content and localisation of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants.

5-Methylcytosine has been found in all pyrimidine isopliths isolated from the DNA of cotton plants, but it localizes predominantly in tri- (about 52%) and dipyrimidine (about 22%) clusters. The 5-methylcytosine distribution by pyrimidine isopliths in DNA of cotton plants is specific and quite different from that in other plant and animal DNA studied. The total 5-methylcytosine content in DNA from wilt-infected cotton plants (2.3 mol %) is less than half that found in DNA from non-infected cotton plants (4.9 mol %). No other visible differences (G+C content, Tm, deltaT, s20,w, frequencies of pyrimidine clusters and others) in these DNA have been found. This suggests that in wilt-infected plants, no essential alteration in DNA sequence or molecular population takes place. As a result of wilt infection 5-methylcytosine completely disappears from dipyrimidine oligonucleotides of cotton plant DNA; its content decreases markedly in long pyrimidine clusters (heptaoligonucleotides and longer) and in C3, C2 T, CT2 fragments. Thus, DNA in wilt-infected plant cells is specifically undermethylated (demethylated). The induced alteration in DNA methylation may be considered one of the possible mechanisms for the specific distortion of gene activity of host cells and primary fungal pathogenic action on plants.     

Distribution of 5-methylcytosine in phage lambda genome methylated by DNA methylase Eco RII

The distribution of 5-methylcytosine in Eco RI-Bam HI fragments of phage lambda DNA in vitro methylated by Eco RII methylase has been studied. The general picture of distribution of methylated sites in phage lambda DNA is slightly different from the statistical distribution. However, the sites have been found, where the distribution of 5-methylcytosine is not accidental. A complete absence of 5-methylcytosine in the J-fragment, a genome lambda area essential for site-specific recombination, has been found. The absence of Eco RII is supposed to be the best protection of this area of phage genome from the increased mutagenesis, characteristic for nucleotide sequences methylated by DNA-methylated Eco RII and Eco RII type.     

DNA methylation in xeroderma pigmentosum

DNA methylation was examined in xeroderma pigmentosum (XP) cells. The amount of 5-methylcytosine (mC) in DNA from XP cells was about 70% of that in DNA from normal controls. Southern hybridization analysis showed that the HLA-DR alpha gene in XP lymphocyte B cells was differently methylated from normals, but its expression was apparently unaffected. The methylation of dihydrofolate reductase, a housekeeping gene, was the same as in controls. The revertants to UV resistance from XP fibroblasts recovered a methylation level close to that of normal cells. Results suggested that XP DNA was undermethylated non-randomly, and that DNA methylation might be associated with DNA repair function.     

5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result.

The 5-methylcytosine content of nuclear DNA from nuclear hepatocellular tissues was determined during various phases of hepatic regeneration and carcinogenesis. DNA from premalignant nodules and primary hepatocellular carcinomas induced by exposure to acetylaminofluorene, as well as PHC induced by diethylnitrosamine was undermethylated by 20%, 45%, and 32.5% respectively. Since a 12.5% hypomethylation occurred during the DNA synthetic phase of hepatic regeneration, the effect of cell proliferation on DNA-methylation in malignancies was examined in transplantable hepatocellular carcinomas. The DNA from two transplantable hepatocellular carcinoma lines was less methylated than predicted rates of cell division in these tumors. This finding suggested that an aberration in endogenous DNA methylation may occur during neoplastic transformation.     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica

The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 µM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired.     

Bovine thymus satellite I DNA sequences, which are highly methylated, are preferentially located in H1-rich chromatin

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5-7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.     

Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells.

Duplex heavy-light (HL) DNAs synthesized in the presence of brdUrd and methylation inhibitors were separated from bulk cellular DNA by CsCl density gradient centrifugation and analysed for 5-methylcytosine (5mC) contents by HPLC. DNAs synthesized in the presence of 5 mM ethionine or 2 mg/ml cycloleucine were not detectably hypomethylated, was undermethylated with respect to control DNA. The heavy, or H-strand, in which up to 5% of the cytosine residues were replaced by intact 5-azacytosine, was undermethylated and the HL duplex DNA was therefore strand asymmetrically methylated. This duplex DNA served as an efficient substrate for a crude DNA methyltransferase preparation which transferred the methyl group from S-adenosylmethionine specifically into cytosine residues within the hypomethylated H strand. Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA may be responsible for the inhibitory effect of 5-azacytidine on DNA methylation.     

S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.     

An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

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.     

Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures

Summary Comparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.     

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.     

Phylogenetic evidence of a role for 5-hydroxymethyluracil-DNA glycosylase in the maintenance of 5-methylcytosine in DNA.

5-Hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of thymine. It is also the product of the deamination of 5-hydroxymethylcytosine (HmCyt) which may be formed via oxidation of 5-methylcytosine (MeCyt). HmUra is removed from DNA by a DNA glycosylase which, together with HmCyt-DNA glycosylase, is unique among DNA repair enzymes in being present in mammalian cells but absent from bacteria and yeast. We found HmUra-DNA glycosylase activity in a wide variety of vertebrate and invertebrate animals (except Drosophila) and in protozoans. In most vertebrate organisms the highest specific activity was in nervous and immune system tissue. The phylogenetic distribution of HmUra-DNA glycosylase correlates with the presence of 5-methylcytosine (MeCyt) as a regulator of gene expression. This distribution of activity supports the contention that HmUra-DNA glycosylase aids in the maintenance of methylated sites in DNA.     

Methylation of nuclear DNA in Physarum polycephalum.

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M--) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.