The sequence specificity of vertebrate DNA methylation.

The relative quantity of 5-methyl cytosine in vertebrate nuclear DNA shows species and tissue variation. To determine whether this is due to the action of species or cell specific DNA methylases the sequence specificity of the 5-methyl cytosine distribution in the DNA of a range of cells has been partially characterised. The pattern of methylation was found to be remarkably constant and indicates stringent evolutionary conservation of the characteristics of vertebrate DNA methylation.     

High sensitivity mapping of methylated cytosines.

An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.     

Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms

In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.     

Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA

The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.     

Region-specific DNA methylation in the preimplantation embryo as a target for genomic plasticity

It has been long known that the unique genetic sequence each embryo inherits is not the sole determinant of phenotype. However, only recently have epigenetic modifications to DNA been implicated in providing potential developmental plasticity to the embryonic and fetal genome, with environmental influences directly altering the epigenetic modifications that contribute to tissue-specific gene regulation. Most is known about the potential environmental regulation of DNA methylation, epigenetic addition of methyl groups to cytosine residues in DNA that acts in the long-term silencing of affected sequences. While most attention has been paid to the methylation of imprinted gene sequences, in terms of developmental plasticity there are many more parts of the genome that are methylated and that could be affected. This review explores the distribution of cytosine methylation in the genome and discusses the potential effects of regional plasticity on subsequent development. Widening our consideration of potentially plastic regions is likely to greatly enhance our understanding of how individuals are shaped not only by DNA sequence, but by the environment in which pluripotent embryonic cells are transformed into the many cell types of the body.     

Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification.

We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.     

Determination of the recognition sites of cytosine DNA-methylases from Escherichia coli SK.

Two different cytosine DNA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5'...NpCpCpApGpGpN...3'. This sequence is identical with the recognition site of the hsp II type determined by RII plasmid but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5' side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GII site: 5'...NpGpGpApCpCpN...3'. In this case cytosine at the 3'-end of the recognition site is methylated.     

Sterol replacement in saccharomyces cerevisiae. Effect on cellular permeability and sensitivity to nystatin

More than 80% of the cellular ergosterol can be replaced by cholesterol in a sterol requiring mutant strain of Saccharomyces cerevisiae. The effect of this replacement, as well as the effect of sterol starvation on the uptake and exit of cytosine and α-aminoisobutyric acid (α-AIBA) was studied in an attempt to elucidate the role of sterols in cellular permeability. Neither the exit of cytosine nor the exit of α-AIBA was affected by changes in the sterol content of the cell. Cells grown on cholesterol or on ergosterol had very similar rates of cytosine uptake, but a lower rate was found for sterol-starved cells. This difference may be a consequence of the cellular growth rate. However, nystatin induces a much slower exit of α-AIBA in cells grown on cholesterol than in cells grown on ergosterol. This strongly suggests that a change in membrane structure has taken place.     

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.     

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.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI.

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI 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 HinfI 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 HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, 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.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Sensitivity of the restriction endonucleases HaeIII, BsrI, EaeI and CfrI to cytosine N4-methylation.

HaeIII, BsrI and NgoII are isochizomers that recognize the sequence GGCC while EaeI and CfrI recognize the overlapping sequence YGGCCR. It has previously been shown that all these enzymes are inhibited by cytosine C5-methylation within the recognition sequence. The methylation sensitivities of these enzymes to cytosine N4-methylation have not been previously reported. In this paper we present data demonstrating that all these enzymes, except NgoII, are inhibited by cytosine N4-methylation of the second 5' cytosine residue within the recognition sequence.     

Arthrobacter viscosus DNA is modified at GATC sequence

Arthrobacter viscosus DNA was resistance to digestion by restriction enzymes that are sensitive to methylation of the cytosine residue (but not of adenine) within the GATC recognition sequence. Restriction enzymes sensitive to methylation of cytosine in other recognition sequences were not affected. A. viscosus DNA thus appeared to contain methylated cytosine specifically at the GATC sequence.     

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.     

Roles,and establishment,maintenance and erasing of the epigenetic cytosine methylation marks in plants

Heritable information in plants consists of genomic information in DNA sequence and epigenetic information superimposed on DNA sequence. The latter is in the form of cytosine methylation at CG, CHG and CHH elements (where H = A, T or C) and a variety of histone modifications in nucleosomes. The epialleles arising from cytosine methylation marks on the nuclear genomic loci have better heritability than the epiallelic variation due to chromatin marks. Phenotypic variation is increased manifold by epiallele comprised methylomes. Plants (angiosperms) have highly conserved genetic mechanisms to establish, maintain or erase cytosine methylation from epialleles. The methylation marks in plants fluctuate according to the cell/tissue/organ in the vegetative and reproductive phases of plant life cycle. They also change according to environment. Epialleles arise by gain or loss of cytosine methylation marks on genes. The changes occur due to the imperfection of the processes that establish and maintain the marks and on account of spontaneous and stress imposed removal of marks. Cytosine methylation pattern acquired in response to abiotic or biotic stress is often inherited over one to several subsequent generations. Cytosine methylation marks affect physiological functions of plants via their effect(s) on gene expression levels. They also repress transposable elements that are abundantly present in plant genomes. The density of their distribution along chromosome lengths affects meiotic recombination rate, while their removal increases mutation rate. Transposon activation due to loss of methylation causes rearrangements such that new gene regulatory networks arise and genes for microRNAs may originate. Cytosine methylation dynamics contribute to evolutionary changes. This review presents and discusses the available evidence on origin, removal and roles of cytosine methylation and on related processes, such as RNA directed DNA methylation, imprinting, paramutation and transgenerational memory in plants.     

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.