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PubMed1985

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗
PubMed1990

Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus.

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.

Amino Acid Sequence↗
PubMed1986

Cloning and characterization of the dcm locus of Escherichia coli K-12.

The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.

Base Sequence↗
PubMed1993

Hypomethylation of DNA: a possible nongenotoxic mechanism underlying the role of cell proliferation in carcinogenesis.

DNA methylation (i.e., the 5-methylcytosin content of DNA) plays a role in the regulation of gene activity. There is a persuasive body of evidence indicating that differential methylation of DNA (i.e., 5-methylcytosine versus cytosine) is a determinant of chromatin structure and that the methyl group provides a chemical signal that is recognized by trans-acting factors that regulate transcription. Hypomethylation (i.e., low levels of DNA 5-methylcytosine) of a gene is necessary but not sufficient for its expression, and, therefore, a hypomethylated gene can be considered to possess an increased potential for expression as compared to a hypermethylated gene. Cell proliferation is a fundamental component of carcinogenesis. It plays a key role in expanding clones of initiated cells and, in addition, cell replication may contribute to carcinogenesis by facilitating mutagenesis. This can occur either by causing the fixation of promutagenic DNA-damage before repair or as a consequence of a "normal" error occurring during DNA replication. During periods of cell proliferation the established pattern of DNA methylation is maintained by the action of a maintenance methylase following DNA replication. Changes in the methylation status of a gene provide a mechanism by which its potential for expression can be altered in an epigenetic heritable manner, and it is expected that modifications in DNA methylation would result from threshold-exhibiting events.

5-Methylcytosine↗
PubMed2000

Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Alkylating Agents↗
PubMed1997

A ColE1-type plasmid from Salmonella enteritidis encodes a DNA cytosine methyltransferase.

The multicopy plasmid pFM366 was isolated from a virulent Salmonella enteritidis strain and was found to code for DNA methylase activity (Ibáñez and Rotger, 1993). The present work was aimed at characterizing the genetic organization and functional features of this 5.6 kb plasmid. We found pFM366 almost identical to the plasmid P4 isolated from Shigella sonnei, that encodes the SsoII restriction-modification system (Karyagina et al., 1993), and related to other ColE1-type plasmids. Examination of these plasmids revealed a common organization which suggests they were the result of similar recombinational events. The cytosine methylase of pFM366 is nearly identical to M. SsoII, whereas the gene encoding the restrictase homologous to R. SsoII is truncated and its product is inactive. The expression of the cytosine methylase encoded by pFM366 is strongly affected by deletion of regions located upstream and downstream of its ORF, and is negatively controlled by the rpoS gene in Escherichia coli. The methylase activity encoded by pFM366 induces the SOS response, which could be responsible for the observed delay in the growth of E. coli.

Amino Acid Sequence↗
PubMed1991

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.

Amino Acid Sequence↗
PubMed2000

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.

5-Methylcytosine↗
PubMed1987

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational.

Bacterial Outer Membrane Proteins↗
PubMed1976

A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA.

Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.

Base Sequence↗
PubMed1988

Isolation and characterization of the modification methylase M . SinI.

A sequence-specific modification methylase (M . SinI) was isolated and purified from Escherichia coli harboring a derivative of recombinant plasmid pSI4 (see accompanying manuscript: C. Karreman and A. de Waard, J. Bacteriol. 170:2527-2532, 1988), which contains a Salmonella infantis DNA insert. The enzyme uniquely methylates the internal deoxycytidylate residue in the nucleotide sequence GG(A/T)MeCC, thereby protecting DNA completely against cleavage by restriction endonuclease R . SinI or R . AvaII [GG(A/T)CC], and in part against cleavage by R . Sau96I (GGNCC).

Base Sequence↗
PubMed1995

Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells.

To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase. Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end-joining or homologous recombination. Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells. These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure.

Animals↗
PubMed1983

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.

Cloning, Molecular↗
PubMed1989

Control of partial digestion combining the enzymes dam methylase and MboI.

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.

DNA↗
PubMed1982

Expression of the Eco RI restriction-modification system and the construction of positive-selection cloning vectors.

The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.

Base Sequence↗
PubMedDate not supplied

[Modification of the modification-restriction system in staphylococci].

A new system of host specificity of DNA, called Sau67 according to the available nomenclature, was identified in Staphylococcus aureus 6782 strain by means of cross titration with staphylophage 729 considering that the phage exhibited the highly effective absorption properties. A total preparation of Sau67 methylases was isolated using ammonium sulfate fractionation. The enzyme preparation contained methylases of cytosine and adenine, where the activity of adenine methylases constituted only for 5% of the total methylase activity. As shown by kinetics of methylation low content of unspecific cellular nucleases was found in the St. aureus 6782 strain; these reasons are important for isolation of restricting endonucleases containing in the strain. 100 micrograms of protein of the total enzymatic fraction enabled to methylate the acceptory DNA at the maximal rate within 1.5 hr of incubation in phosphate buffer, pH 7.9. The fraction of cytosine methylases free of adenine methylating activity was obtained after chromatography on Sepharose blue with NaCl concentration stepwise gradient.

Chromatography, Agarose↗
PubMed2001

Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products.

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.

Amino Acid Sequence↗