Restriction and modification in Bacillus subtilis: sequence specificities of restriction/modification systems BsuM, BsuE, and BsuF.

The sequence specificities of three Bacillus subtilis restriction/modification systems were established: (i) BsuM (CTCGAG), an isoschizomer to XhoI; (ii) BsuE (CGCG), an isoschizomer to FnuDII; and (iii) BsuF (CCGG), an isoschizomer to MspI, HpaII. The BsuM modification enzyme methylates the 3' cytosine of the recognition sequence. The BsuF modification enzyme methylates the 5' cytosine of the sequence, rendering such sites resistant to MspI degradation and leaving the majority of sites sensitive to HpaII degradation.     

Effect of CpG methylation on gene expression in transfected plant protoplasts

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.     

DNA methylation diminishes bleomycin-mediated strand scission

Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.     

Changes in methylation of tissue cultured soybean cells detected by digestion with the restriction enzymes HpaII and MspI

Each of the tandemly arranged 5S RNA genes of soybean contain two CCGG sites which, if unmethylated, can be digested by both MspI and HpaII. Methylation of the internal cytosine (CmeCGG) prevents digestion by HpaII but allows digestions by MspI.Suspension cultures were prepared from soybean plants and the DNA from these cultures was examined for the susceptibility of 5S RNA genes to digestion by MspI and HpaII. 5S genes from DNA extracted from intact plants can be partially digested with MspI but not at all by HpaII. In contrast, shortly after cells were cultured the 5S RNA could be hydrolyzed by both HpaII and MspI. After prolonged cell culture, the 5S genes from some cell lines were found to have become partially or even completely resistant to HpaII digestion. The results suggest that lack of methylation can occur when cells are cultured and that such methylation may play a role in the heritable changes observed in cell culture.Research supported by Grant 01498 from the National Institutes of Environmental Health Sciences     

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

Bacillus subtilis phage SPR codes for a DNA methyltransferase with triple sequence specificity.

SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated. We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI. This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase. SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively. These mutants have not lost their ability to methylate CC(A/T)GG sites. Mutation SPR26 has lost the ability to methylate all three sites. Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.     

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.     

The somatic replication of DNA methylation

We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.     

The effects of 5-azacytidine on the expression of the rat growth hormone gene. Methylation modulates but does not control growth hormone gene activity

The role of DNA methylation in the expression of the rat growth hormone (rGH) gene was assessed by using a hypomethylating agent, 5-azacytidine, and the iso-schizomeric restriction enzymes MspI and HpaII. 5-Azacytidine increased rGH mRNA 3-8-fold in GH3D6 cells, a subclone of rat pituitary tumor cell lines that expresses one-tenth to one-fifteenth the GH expressed by two other clones, GH3 and GC. The effect was also detected at the level of pre-mRNA. The effect was independent of glucocorticoids and thyroid hormones and was found to be inheritable. The DNA methylation pattern generated by the isoschizomeric restriction enzymes indicated that the HpaII sites in the rGH gene were mostly methylated in GH3D6 cells but mostly unmethylated in GC cells. After treatment with 5-azacytidine, about 22% of these HpaII sites in GH3D6 cells became unmethylated. Thus, DNA methylation correlates inversely with the expression of the rGH gene in these cell lines. However, three other observations indicate that factors in addition to DNA methylation control rGH expression. First, in GC cells, even though most of the HpaII sites are unmethylated, the gene is not fully expressed. Second, in rat hepatoma cells, which do not express GH at all, the GH gene is less methylated than that in GH3D6 cells. Third, within the sensitivities of the assay methods, 5-azacytidine has no effect on the GH gene when it is completely silent. Taken together, the findings indicate that DNA methylation modulates but does not control GH gene expression. It is tempting to speculate that DNA methylation can influence expression only when the gene is committed to express.     

Unmethylated domains in vertebrate DNA.

We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA.     

The sequence GGCmCGG is resistant to MspI cleavage.

MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells.     

Variable methylation of the ribosomal RNA genes of the rat.

Both the pattern and level of rRNA gene methylation vary in the rat. This variation reflects stages in the maturation process and perhaps the level of gene expression in different tissues. We studied methylation at a common site, the inner cytosine of the sequence CCGG, by hybridizing 32P-rRNA to DNA digests obtained with endonuclease Msp I (which cleaves CCGG and CMCGG) and its isochizomer, HpaII (which cleaves only CCGG). In the liver, the changing pattern of rRNA gene methylation reflected the late stages of development: the rRNA genes were mostly unmethylated at 14 days gestation; by 18 days gestation, about 30% of them were methylated, and this level persisted into adulthood. In 18-day DNA, the methylation was uniform, but in adult DNA, the methylation pattern was discontinuous, because otherwise methylated genes contained a demethylated region. Similar developmental changes were observed in brain DNA. In a tissue culture cell line, the change from the continuous to the discontinuous pattern of the methylation could be induced by transformation with Kirsten sarcoma virus. And, in adult tissues, the lowest level of rRNA gene methylation was found in rapidly growing jejunal epithelium, and the highest level, in non-growing spermatozoa.     

Topographical distribution of 5-methylcytosine in animal and plant DNA.

The topographical distribution of 5-methylcytosine on animal and plant cell DNA has been examined with methyl-sensitive restriction enzymes and gel electrophoresis analysis. These DNAs digested with the enzyme HpaII have a partially bimodal size distribution, indicating the existence of clusters of methylated and unmethylated CCGG sites in the DNA. By analyzing the methylation state of all CG moieties in restricted DNA fractions, it was possible to show that these genomes are, in general, arranged as clusters of relatively highly methylated and undermethylated regions. Plant DNA also contains 5-methylcytosine in the prototype sequence C-X-G. Restriction of this DNA with EcoRII revealed that these methyl groups are also distributed in clusters, suggesting that this is a general phenomenon. The undermethylated areas may correspond to the active fraction of the genome.     

Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.     

Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.     

MspI, an isoschizomer of hpaII which cleaves both unmethylated and methylated hpaII sites.

The cleavage of DNA by restriction endonucleases HpaII and HapII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII available from New England Biolabs, cleaves DNA irrespective of the presence of a methyl group at this position. This enzyme cleaves DNA from Haemophilus parainfluenzae and Haemophilus aphrophilus readily while HpaII and HapII cannot degrade these DNAs. Practically all HpaII sites in mammalian sperm DNA are also protected by methylation at the internal C position since HpaII and HapII barely cleave this DNA (average molecular weight 40 kb). MspI, however, cleaves the DNA to an average size of about 5 kb.     

In vitro methylation of DNA with Hpa II methylase.

The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA.     

Non-uniform distribution of methylatable CCGG sequences on human chromosomes as shown by in situ methylation

We carried out in situ methylation of human chromosomes with the HpaII methylase using [³H]methyl-S-adenosyl-l-methionine as the methyl group carrier. Autoradiographs localising [³H]methyl groups show methylatable CCGG sequences in the R-bands as well as in the short arms of the acrocentric chromosomes that include ribosomal DNA. The strongest labelling was observed over a subset of R-bands, including T-bands. Since methylatable CCGG sequences are representative of the unmethylated fraction of DNA, we suggest that differences in the degree of DNA methylation could be involved in the structure and function of chromosomal bands.