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PubMed1982

In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells.

The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low frequency of reversion to the aprt+ phenotype was observed. In all of these clones, this reversion was accompanied by reorganization and undermethylation of the aprt sequences. These results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.

5-Methylcytosine↗

PubMed1993

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Cloning, Molecular↗

PubMed1990

Characterization of the genetic determinants of SsoII-restriction endonuclease and modification methyltransferase.

The genes encoding SsoI and SsoII restriction endonuclease (ENase) and methyltransferase (MTase) are located on the small plasmids P6 and P4, respectively, of Shigella sonnei strain 47. Functions provided by plasmids P5, P7 and P9, which include colicinogenicity and immunity to colicin E1, resistance to streptomycin (Sm), and conjugative DNA transfer, respectively, have also been identified. The genes of the SsoII restriction-modification (R-M) system have been cloned into Escherichia coli expressing the 35-kDa (ENase) and 43-kDa (MTase) products. A restriction map of the P4 plasmid DNA was determined, and the approximate location of the genes encoding SsoII ENase and MTase (ssoIIR and ssoIIM) on that have been established. SsoI is an isoschisomer of EcoRI and SsoII cleaves the 5'-/CCNGG/recognition sequence producing 5'-protruding 5-nt long cohesive ends.

Cloning, Molecular↗

PubMed2004

Lactococcal plasmid pNP40 encodes a novel, temperature-sensitive restriction-modification system.

A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis. The system comprises four adjacent similarly oriented genes that are predicted to encode two m(5)C methylases and two restriction endonucleases. The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species. This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19 degrees C. A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype. A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperature-sensitive profile. This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.

Bacteriophages↗

PubMed2005

Study of tissue-specific CpG methylation of DNA in extended genomic loci.

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.

Animals↗

PubMed1988

The cloning, purification and characterization of the Eco RV modification methylase.

The gene for the Eco RV methylase has been cloned into a plasmid under control of the strong lambda PL promoter and overexpressed in E. coli. This plasmid, pVIC1, gives reliable overexpression of the methylase at levels of about 20% of total protein. Maximum yields of soluble protein are achieved after about 6 hours of induction. If the cells are harvested later than this much of the enzyme is found in the pellet fraction following centrifugation. A two column purification scheme using phosphocellulose and Blue-Sepharose chromatography has been developed. This yielded pure methylase in amounts of 5mg per gram E. coli cell paste. The enzyme is monomeric and methylates the first deoxyadenosine residue in its recognition sequence GATATC.

Base Sequence↗

PubMed2000

LldI, a plasmid-encoded type I restriction and modification system in Lactococcus lactis.

A plasmid-encoded type I restriction and modification (R-M) system, designated LldI, was identified in Lactococcus lactis biovar diacetylactis LD10-1. LldI consists of three genes encoding endonuclease, methylase and specificity subunits, respectively. RT-PCR analysis revealed that the three genes are co-transcribed as a polycistronic mRNA in L. lactis. The specificity subunit of LldI differs significantly in the target recognition domains from those of other type I R-M systems, suggesting that LldI confers a novel specificity in L. lactis.

Amino Acid Sequence↗

PubMed2003

Epigenetic transitions: towards therapeutic targets.

Therapeutic approaches aimed at developing epigenetically-effective drugs are under intense investigation. Several classes of enzymes regulating histone acetylation and DNA methylation, which are required for epigenetic transitions, offer attractive targets for therapeutic interventions. Imbalances in histone acetylation and DNA methylation may play a significant role in the development of cancer and leukaemia and may provide a mechanistic rationale for targeting epigenetic modifications. Clinical trials designed to evaluate inhibitors of DNA methylation and histone deacetylase inhibitors are showing encouraging results in cancer patients. A growing quantity of data from preclinical research supports the notion that epigenetically-effective drugs could also find an application in other therapeutic areas. A number of emerging biomarkers may prove useful for monitoring drug effects and defining molecular signatures of response, toxicity and effective dose.

Acetylation↗

PubMed1997

Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis.

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.

Amino Acid Sequence↗

PubMed1984

Selective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA.

The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R . BsuRI and its cognate methylase even though greater than 20 copies of the target sequence, 5' ... GGCC ... 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' ... GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5' ... GGCCG ... 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5' ... GCNGC ... 3' (R . Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.

Bacillus subtilis↗

PubMed1982

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes.

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.

Azacitidine↗

PubMed1999

Molecular mechanisms of resistance to commonly used non-betalactam drugs in Streptococcus pneumoniae.

This article reviews the molecular mechanisms of resistance to fluoroquinolones, erythromycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole in Streptococcus pneumoniae. Resistance to fluoroquinolones primarily involves mutations in the DNA gyrase gene, gyrA, and in the topoisomerase IV genes, parC and parE, although in vitro studies have indicated that some strains may use an efflux mechanism for resistance to certain fluoroquinolones. Ciprofloxacin resistance results from initial and necessary mutations in ParC leading to low-level resistance and subsequent mutations in GyrA leading to high-level resistance. Sparfloxacin resistance results from initial mutations in GyrA, with ParC mutations occurring subsequently. A single amino acid substitution in ParE has also been associated with low-level resistance in S pneumoniae. Two mechanisms have been described for resistance to erythromycin. Coresistance to macrolides, lincosamides, and streptogramin B type antibiotics is a result of modification of the ribosome through methylation of an adenine residue in domain V of the 23S rRNA. This methylation is encoded by the methylase gene, ermAM. Resistance only to 14-and 15-membered macrolides is a result of efflux of the antibiotic from the cell, encoded by the gene, mefE, in S pneumoniae, and appears to be rapidly emerging as the predominant mechanism of resistance to erythromycin in many countries. The production of chloramphenicol acetyltransferase, an enzyme capable of catalyzing the conversion of chloramphenicol to its nonfunctional 1-acetoxy, 3-acetoxy, and 1,3-diacetoxy derivatives, leads to chloramphenicol resistance in S pneumoniae. Chloramphenicol acetyltransferase is encoded by a cat gene identical to the cat gene from the Staphylococcus aureus plasmid, pC194. Tetracycline resistance occurs through ribosomal protection encoded by the genes tet(M) and tet(O). It is possible that the Tet(M) and Tet(O) proteins cause tetracycline to be released from the ribosome, although the precise mechanism remains unclear. Resistance to trimethoprim is mediated through a single amino acid substitution in the chromosomal dihydrofolate reductase gene of S pneumoniae, which is thought to disrupt the bond with trimethoprim without affecting the action of the dihydrofolate reductase. Sulphonamide resistance appears to result from repetitions of one or two amino acids in the chromosomal dihydropteroate synthase. Although resistance exists to nearly all antimicrobial agents used in the treatment of S pneumoniae infections, ongoing research into new or alternative therapies is encouraging.

Animals↗

PubMed2004

Expression analysis of the epigenetic methyltransferases and methyl-CpG binding protein families in the normal B-cell and B-cell chronic lymphocytic leukemia (CLL).

The importance of epigenetic modifications in carcinogenesis has been a source of controversy for some time. There is little doubt that changes in genomic hypermethylation contribute to the silencing of tumor suppressor genes. Furthermore, recent studies have also identified the significance of genomic hypomethylation associated with chromosomal instability and tumorigenesis. One of the most perplexing questions regarding epigenetic modifications and leukemogenesis is the relationship with DNA methyltransferases (DNMT's). The primary function of the DNMT enzymes is to methylate genomic DNA, whereas the methyl-CpG binding domain proteins (MBD) interpret this methylation signal and regulate gene expression and chromatin behavior. In this study we analyse these gene families by quantitative real-time PCR to investigate whether expression levels and the B-cell chronic lymphocytic leukemia (B-CLL) phenotype are associated. Furthermore, given the epigenetic crosstalk between genome stability and the histone chromatin code we have analysed eukaryotic histone methyltransferase (Eu-HMTaseI). Surprisingly, we did not observe significant changes in DNMT1 expression in B-CLL cases when compared to normal lymphocytes, regardless of whether we normalise against GAPDH or PCNA as reference standards. Indeed, expression of the maintenance and de novo methylases were independently regulated. Of particular note was the significant down regulation of DNMT3b. Furthermore, we observed a positive correlation between HMTaseI expression levels and stage of leukemia suggesting that changes in the methylation patterns in B-CLL may represent deregulation of the epigenetic repertoire that also include the methylation dependent binding proteins, MBD2 and MeCP2. We envisage changes in the epigenetic program are multifactorial in nature and postulate that the prevalent genomic methylases just one component of a larger epigenetic repertoire.

B-Lymphocytes↗

PubMed1988

Cloning of the HhaI and HinPI restriction-modification systems.

The genes for the HhaI (Roberts et al., 1976) and HinPI (Roberts, 1987) restriction-modification (R-M) systems have been cloned in pBR322. The HhaI system was isolated on a 9-kb PstI fragment, and the HinPI system was isolated on two PstI fragments of 1.5 and 4.6 kb in length. The clones were isolated by selecting for recombinant molecules that had protectively modified themselves. The HhaI and HinPI R-M systems recognize the same sequence, GCGC, but hybridization between the DNA fragments encoding them does not take place.

Bacterial Proteins↗

PubMed1988

Cytosine methylation as an effector of right-handed to left-handed DNA structural transitions.

Cytosine methylation has energetic and structural influences on left-handed Z-DNA formation in supercoiled plasmids. The restriction and modification enzymes from Haemophilus haemolyticus (HhaI and M.HhaI) provide a system to locate and analyze small segments of Z-DNA in large supercoiled plasmids. An approach is outlined that uses M.HhaI as an in vivo conformational probe for the detection of unusual DNA structures in a living cell. Also, characteristic features of the M.HhaI gene and protein are discussed.

5-Methylcytosine↗

PubMed2001

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/.

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Adenosine↗

PubMed1987

High level expression of the EcoP1 modification methylase gene and characterisation of the gene product.

We have cloned the gene coding for the EcoP1 modification methylase in an expression system based on the phage lambda pL promoter and the cI857-coded thermoinducible repressor. We have used this system to purify the enzyme on the 20-30-mg scale and have examined some of its enzymatic properties. The enzyme is a tetramer of Mr 72,000 subunits and is approx. 40% alpha-helical. Experiments with the methyl donor, S-adenosyl methionine, radioactively labelled in different positions indicate that a methyl group is transferred to the enzyme during the reaction in what is most likely a covalent bond.

Cloning, Molecular↗

PubMed1984

Isolation of gram quantities of EcoRI restriction and modification enzymes from an overproducing strain.

Structural genes for EcoRI restriction endonuclease and modification methylase have been inserted into the plasmid vector pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature (Lond.) 292, 128-132) downstream from the bacteriophage lambda pL promoter. Upon induction of pL expression in strains producing a thermolabile lambda cI857 repressor, synthesis of EcoRI polypeptides is enhanced to the extent that after 4 h they represent several per cent of the total cell protein. Purification of activities overproduced in this manner yields preparations of endonuclease and methylase which appear identical to those obtained from conventional sources, with overall yields corresponding to 0.5 to 0.9 g of each enzyme/kg of cell paste.

DNA Restriction Enzymes↗