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PubMed2007

Colorectal serrated adenocarcinoma.

Colorectal cancer (CRC) ranks among the three most common cancers in terms of both cancer incidence and cancer-related deaths in most Western countries. Serrated adenocarcinoma is a recently described, distinct variant of CRC, accounting for about 7.5% of all CRCs and up to 17.5% of most proximal CRCs. It has been postulated that about 10-15% of sporadic CRCs would have their origin in serrated polyps that harbour a significant malignant potential. These lesions include hyperplastic-type aberrant crypt foci, hyperplastic polyps, sessile serrated adenomas, admixed polyps and serrated adenomas, and constitute the so-called 'serrated pathway', which is distinct from both the conventional adenoma-carcinoma pathway and the mutator pathway of hereditary non-polyposis CRC and is characterized by early involvement of oncogenic BRAF mutations, excess CpG island methylation (CIM) and subsequent low- or high-level DNA microsatellite instability (MSI). Methylation of hMLH1 is likely to explain the increased frequency of high-level MSI (16%) and methylation of MGMT is postulated to explain the low-level MSI (29%) in serrated adenocarcinomas. Reproducible histopathological criteria for serrated adenocarcinoma have recently been established and they have been qualified by DNA expression analysis for 7928 genes, showing clustering of serrated adenocarcinomas into a molecular entity apart from conventional adenocarcinoma, and representing with distinct down-regulation of EPHB2, PTCH and up-regulation of HIF1alpha.

Adaptor Proteins, Signal Transducing↗
PubMed1992

The suicidal DNA repair methyltransferases of microbes.

Virtually every organism so far tested has been found to possess an extremely efficient DNA repair mechanism to ensure that certain alkylated oxygens do not accumulate in the genome. The repair is executed by DNA methyltransferases (MTases) which repair DNA O6-methylguanine (O6MeG), O4-methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repair of just one, or sometimes two, O-alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection against death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well-characterized microbial DNA repair MTases.

Amino Acid Sequence↗
PubMed1995

Overexpression of DNA methyltransferase in myoblast cells accelerates myotube formation.

We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.

Animals↗
PubMed1993

Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli.

We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis, and detected the presence of small plasmids (3-5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid. A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning. The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S. enteritidis, and with another small plasmid from Salmonella typhimurium. A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb PvuII restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.

Blotting, Southern↗
PubMed2001

DNA methylation and epigenetic inheritance in plants and filamentous fungi.

Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.

DNA Methylation↗
PubMed2006

Identification of a DNA methyltransferase gene carried on a pathogenicity island-like element (VPAI) in Vibrio parahaemolyticus and its prevalence among clinical and environmental isolates.

In this study we identified a putative virulence-associated DNA methyltransferase (MTase) gene carried on a novel 22.79-kb pathogenicity island-like element (VPAI) in V. parahaemolyticus. The V. parahaemolyticus MTase gene was shown by PCR to be prevalent (>98%) in pandemic thermostable direct hemolysin gene-positive isolates, which suggests that VPAI may confer unique virulence traits to pandemic strains of V. parahaemolyticus.

Amino Acid Sequence↗
PubMed2006

M.SpyI, a DNA methyltransferase encoded on a mefA chimeric element, modifies the genome of Streptococcus pyogenes.

While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Phi10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Phi10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Phi10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5'-C(m)CNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Phi10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Phi10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.

Alleles↗
PubMed1991

Nucleotide sequence of the gene (ard) encoding the antirestriction protein of plasmid colIb-P9.

The IncI1 plasmid ColIb-P9 was found to encode an antirestriction function. The relevant gene, ard (alleviation of restriction of DNA), maps about 5 kb from the origin of transfer, in the region transferred early during bacterial conjugation. Ard inhibits both restriction and modification by each of the four type I systems of Escherichia coli tested, but it had no effect on restriction by either EcoRI, a type II system, or EcoP1, a type III system. The nucleotide sequence of the ColIb ard gene was determined; the predicted molecular weight of the Ard polypeptide is 19,193. The proposed polypeptide chain contains an excess of 25 negatively charged amino acids, suggesting that its overall character is very acidic. Deletion analysis of the gene revealed that the Ard protein contained a distinct functional domain located in the COOH-terminal half of the polypeptide. We suggest that the biological role of the ColIb Ard protein is associated with overcoming host-controlled restriction during bacterial conjugation.

Amino Acid Sequence↗
PubMed1991

Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA.

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.

Conjugation, Genetic↗
PubMed1992

Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.

Chromosome Mapping↗
PubMed1994

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.

Alkylating Agents↗
PubMed1994

Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis.

The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis. Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site. Disruption of the hip locus increased activity from the fusion 16-fold. Repression was restored by supplying HipB in trans. HipB was overexpressed and purified. On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies. HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous and appears to be cooperative. Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB. Strains disrupted in hipB but not in hipA could not be recovered. Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB. HipA is found exclusively in a tight complex with HipB. Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB.

Bacterial Proteins↗
PubMed1995

Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy.

The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3' to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M. LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M. Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M. LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

Amino Acid Sequence↗
PubMed2001

Specificities of eleven different DNA methyltransferases of Helicobacter pylori strain 26695.

Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of Helicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m(4)C, m(5)C, and m(6)A.

Cloning, Molecular↗
PubMed1993

Patterns of frog virus 3 DNA methylation and DNA methyltransferase activity in nuclei of infected cells.

The iridovirus frog virus 3 (FV3) can replicate in culture in fat head minnow (FHM) fish cells or in BHK-21 hamster cells. Viral DNA replication commences about 3 h after infection of FHM cells with FV3. Between 3 and 6 h postinfection (p.i.), a portion of the intranuclear FV3 DNA is partly unmethylated. At later times, p.i., all of the viral DNA in the nuclear and cytoplasmic compartments is methylated at the 5'-CCGG-3' sequences. Cytoplasmic FV3 DNA has not been found unmethylated. We have cloned viral DNA fragments from methylated virion DNA. By using the genomic sequencing technique, it has been demonstrated for segments of the FV3 DNA replicated both in FHM fish and BHK21 hamster cells that in a stretch encompassing a total of 350 bp, all of the analyzed 5'-CG-3' dinucleotides are methylated. The modified nucleotide 5-methyldeoxycytidine is present exclusively in the 5'-CG-3' dinucleotide combination. In the cloned FV3 DNA fragment p21A, an open reading frame has been located. The 5' region of this presumptive viral gene is also methylated in all 5'-CG-3' positions. DNA methyltransferase activity has been detected in the nuclei of FV3-infected FHM cells at 4, 11, and 20 h p.i. In the cytoplasmic fraction, comparable activity has not been observed. These data are consistent with the interpretation that FV3 DNA is newly synthesized and de novo methylated in the nuclei of infected FHM cells and subsequently exported into the cytoplasm for viral assembly.

Amino Acid Sequence↗
PubMed1993

cis regulation of the keratin 18 gene in transgenic mice.

The gene coding for human keratin 18 (K18), a type I intermediate filament protein found in a variety of simple epithelia, is regulated correctly in transgenic mice but is promiscuously expressed after direct transfection into cell culture lines. We have begun an investigation of the mechanisms responsible for the correct regulation of K18 with a comparison of the chromatin state of K18 in permissive and nonpermissive transgenic mouse tissues to identify seven expression-specific, DNase-hypersensitive sites that correlate with known or potential regulatory regions of the gene. Four of these sites are associated with the proximal promoter region and the first intron that has been implicated previously in the transcriptional control of K18. Two hypersensitive sites are associated with a conserved Alu repetitive sequence located immediately upstream of the proximal promoter elements. Transcription of this Alu element in a direction opposite that of K18 was correlated with K18 expression in transgenic tissues. The final hypersensitive site was mapped to exon 6. The potential importance of this region for the expression of K18 was supported by the results of transient expression of the gene and various deleted constructions. In addition, exon 6 and the intron 1 regulatory region were distinguished from the remainder of K18 by differential DNA methylation in expressing and nonexpressing tissues. The CpG-rich proximal promoter and first exon regions remain unmethylated in both permissive and nonpermissive tissues. These results suggest that DNA methylation is not the primary mechanism of control of the gene. An Alu RNA polymerase III transcription unit and exon 6 are implicated in regulation of K18.

Amino Acid Sequence↗