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.     

Specific protection of methylated CpGs in mammalian nuclei

We have compared nuclear accessibility of methylated and nonmethylated sequences using restriction enzymes. MspI, which cuts CpG sites in naked DNA regardless of methylation, cut DNA in intact mouse liver or brain nuclei almost exclusively at CpG islands. Bulk chromatin was not significantly cleaved by MspI but was cleaved extensively by enzymes that do not recognize CpG. Quantitative analysis of limit digests showed that MspI and another methyl-CpG insensitive enzyme, Tth, have a strong bias against cutting methylated sites in these nuclei. Southern analysis confirmed this at three genomic loci. Our results suggest that resistance to nucleases is mediated by factors that are bound specifically to methylated CpGs. MeCP, a protein that binds to methylated DNA in vitro, may be one such factor, since nuclease resistance was significantly reduced in an MeCP-deficient cell line.     

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.     

Methylation and expression of the human thyroglobulin gene

The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene.     

Tissue- and cell-specific casein gene expression. II. Relationship to site-specific DNA methylation

The relationship between DNA methylation and the expression of the gamma- and beta-casein genes was investigated in both expressing and nonexpressing tissues and in isolated tumor cell subpopulations displaying differential casein gene expression. MspI/HpaII digestions of DNA isolated from liver, a totally nonexpressing tissue, indicated that specific sites of hypermethylation existed in these genes as compared to the DNA isolated from casein-producing lactating mammary gland. The positions of these sites were mapped in the gamma-casein gene by comparing total genomic DNA Southern blots to the restriction digests of several overlapping phage clones constituting the gamma-casein gene. In contrast, the methylation status of the HhaI sites in the gamma-casein gene was found to be invariant regardless of the expression status of the gene. The inverse correlation between the hypermethylation of certain MspI/HpaII restriction sites in the casein genes and their potential expressibility was further substantiated by studies in 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary carcinomas, which have an attenuated casein gene expression, and in cell subpopulations isolated from the 7,12-dimethylbenz(a)-anthracene tumor which were either depleted or enriched in casein-producing cells. Analysis of total tumor DNAs indicated that the casein genes were hypermethylated at the same sites observed in liver. However, a very faint hybridization signal was observed in the HpaII digests, suggesting cell-specific methylation differences. We have confirmed the hypomethylation of at least two of these MspI/HpaII sites within the subpopulation containing the casein-producing cells at a level consistent with the relative enrichment in that fraction. These results demonstrate differential site-specific casein gene methylation not only between tissues but also between cell subpopulations within a single tissue.     

A locus on chromosome 11p with multiple restriction site polymorphisms.

We have discovered and characterized a new polymorphic locus on chromosome 11p, D11S12, defined by an arbitrary genomic DNA segment cloned in the plasmid pADJ762. Four different polymorphic restriction sites with minor allele frequencies greater than 5% are revealed by Southern hybridization of this probe and its derivatives to digests of human DNAs. These include two MspI sites, a TaqI site, and a BclI site. The frequencies of the common haplotypes at this locus have been determined in a Utah population. Significant linkage disequilibrium has been demonstrated to exist between some pairs of polymorphic sites. A molecular map of this region has been determined, and the polymorphic sites have been localized. Comparison of physical separation with degree of linkage disequilibrium reveals an interesting case where an MspI site and a TaqI site that are separated by 6.8 kilobases (kb) show a greater degree of disequilibrium with each other than they do with two polymorphic sites located between them. One of the two interior sites is a BclI site that is approximately 0.2 kb away from the TaqI site but shows the same degree of disequilibrium with the TaqI site as with the MspI site 6.7 kb away. Although there is significant linkage disequilibrium at this locus, there are four major haplotypes with frequencies of 5% or greater, and the polymorphic information content (PIC) of this locus is .64.     

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     

The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.     

DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of DNA at seven loci of the nuclear genome]

Seven DNA markers from five genes and one chromosomal region were analysed in Mongolian population using the polymerase chain reaction. The frequencies of alleles of the polymorphisms detected with HindIII in the HBG-2, AvaII in the HBB, MspI and XbaI in the Apo-B, PstI in the D7S8, HincII in the LDLR and allele frequency of the minisatellite fragment in the AT-3 have been determined. The results of the RELP for Apo-B(MspI), LDLR, D7S8 and AT-3 are obtained for the first time among Mongoloids. DNA markers studied demonstrated high level of polymorphisms in the population of Mongolia, except for XbaI and MspI restriction sites at the Apo-B locus. The data obtained for Mongolian population and the literature data were compared.     

Different methylation pattern of melon satellite DNA sequences in hypocotyl and callus tissues

Satellite DNA sequences from Cucumis melo have been examined with respect to modification at CCGG sequences in hypocotyls and in callus tissues. For this purpose, restriction fragments given by HpaII and MspI were compared (both enzymes recognize CCGG sequences but have different sensitivity to methylation at this site). Whereas the methylation level of satellite DNA sequences is on average higher in hypocotyls than in callus tissues, the comparison of partially methylated repeat units of satellite DNA reveals that in callus tissues, all methylated restriction sites are doubly methylated.     

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.     

Methylation of repetitive DNA sequences in the brain during aging of the rat

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15- and 88-week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end-labelling. mCpG doublets are present in two RDS of approximately 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated mCpC doublets also occur. Both mCpG and mCpC doublets are found more in the old than in the young. This age-related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.     

Methylation pattern of mouse mitochondrial DNA.

The pattern of methylation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (m5Cyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methylation was further investigated by determining the state of methylation of several MspI (HpaII) sites. Different sites were found to be methylated to a different extent, implying that methylation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methylation (CpG methylation, partial methylation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes.     

DNA methylation at a CCGG sequence in the large intron of the rabbit β-globin gene: tissue-specific variations

We have analysed DNA modification in a HapII site (CCGG) present in the major intron of the discontinuous rabbit β-globin gene. In most somatic tissues, including erythroid and non-erythroid tissues, about 50% of the DNA is resistant to cleavage at this site by HapII, though 100% cleavage is found with the isoschizomer MspI. Since the former enzyme is unable to cleave CCGG sites if the internal C residue is 5-methyl C (and since methylation is the only form of CpG modification documented in animal DNA), while the latter enzyme cleaves DNA irrespective of methylation at this residue, we infer that 50% of the CCGG sites in the β-globin gene intron are methylated in these tissues.     

Alterations in c-abl gene methylation in cells transformed by phagocyte-generated oxidants

DNA from 10T1/2 cells transformed by activated neutrophils was analyzed for restriction length polymorphisms (RFLPs) in cellular homologues of retroviral oncogenes, and consistent RFLPs were found in MspI sites of the c-abl gene of all PMN-transformed cell lines. MspI digests probed with c-myc, v-Ki-ras, v-Ha-ras or v-mos showed no RFLPs, and none were observed in EcoRI, PstI, HindIII, BamHI, SmaI, Sau3a, MboI, HhaI, or TaqI digests probed with v-abl. Analysis of HpaII digests supports the conclusion that c-abl RFLPs result from differential methylation of the CCGG HpaII/MspI recognition sequence. MspI RFLPs in the c-abl gene may provide markers for oxidant-related genetic injury.     

The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity.

DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures. Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus. Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245-287) to the active site of human DNA ligase I. To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB-truncated protein has a molecular mass of approximately equal 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted. Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.     

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.