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The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure H D underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO 2 )-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.

DNA methylation mediated by DNA methyltransferase (MTase) enzyme is internal cell mechanism which regulate the expression or suppression of crucial genes involve in cancer early diagnosis. Herein, highly sensitive fluorescence biosensing platform was developed for monitoring of DNA Dam MTase enzyme activity and inhibition based on fluorescence signal on mechanism. The specific Au NP functionalized oligonucleotide probe with overhang end as a template for the synthesis of fluorescent silver nanoclusters (Ag NCs) was designed to provide the FRET occurrence. Following, methylation and cleavage processes by Dam MTAse and DpnI enzymes respectively at specific probe recognition site could resulted to release of AgNCs synthesizer DNA fragment and returned the platform to fluorescence signal-on state through interrupting in FRET. Subsequently, amplified fluorescence emission signals of Ag NCs showed increasing linear relationship with amount of Dam MTase enzyme at the range of 0.1-20 U/mL and the detection limit was estimated at 0.05 U/mL. Superior selectivity of experiment was illustrated among other tested MTase and restriction enzymes due to the specific recognition of MTase toward its substrate. Furthermore, the inhibition effect of applied Dam MTase drug inhibitors screened and evaluated with satisfactory results which would be helpful for discovery of antimicrobial drugs. The real sample assay also showed the applicability of proposed method in human serum condition. This novel strategy presented an efficient and cost effective platform for sensitive monitoring of DNA MTase activity and inhibition which illustrated its great potential for further application in medical diagnosis and drug discovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this work, an antifouling electrochemiluminescent (ECL) ratiometric biosensor is designed for the accurate, selective and sensitive detection of DNA methyltransferase (MTase) activity based on a dual-signaling strategy. Briefly, an ITO electrode is used to construct the anti-fouling interfaces with the modification of polyaniline (PANI), AuNPs and peptide. Hairpin DNA molecules containing the symmetric sequence of 5'-CATC-3' are attached onto the modified ITO electrode and the ds-DNA can be cut off in the presence of Dam MTase and DpnI. The residual DNA and two hairpin DNA could lead to the extension of ds-DNA due to the Hybridization Chain Reaction (HCR). ECL signal is amplified significantly with the insertion of PTC-NH 2 molecules into the dsDNA grooves. The ECL PTC-NH2 /ECL Au@luminol is found in a logarithmic linear relation with the concentration of Dam MTase. Moreover, owing to the presence of antifouling peptide on the sensing interface, the ECL biosensor was capable of sensing MTase activity in complex biological media, such as FBS samples and human serum with significantly reduced nonspecific adsorption effect. Assaying Dam MTase in complex sample mixture containing 5% calf serum and 5% human serum further proved the feasibility of this ECL biosensor for early clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Photoelectrochemical (PEC) methods have recently witnessed ever expanding application in bioanalysis, but it is still desirable to further simplify the sensing procedures and develop simple and reliable PEC biosensing approaches. Herein, we proposed a truly label-free and immobilization-free PEC sensing platform, utilizing solution-phase methylene blue (MB) as the signal probe, and bare indium tin oxide (ITO) glass as the photoelectrode. Based on the diffusivity difference between free MB molecules and MB intercalated in DNA G-quadruplex, the activity and inhibition of DNA adenine methyltransferase (Dam), a proof-of-concept methyltransferase (MTase), is quantitatively analyzed. By taking advantage of the endonuclease-catalyzed cleavage of the Dam-methylated hairpin DNA probe, as well as the KF polymerase/Nt.AlwI endonuclease-aided signal amplification, highly sensitive and specific PEC detection of Dam activity has been achieved. Moreover, this approach can be easily extended to assay other types of MTase by choosing the appropriate methylation-sensitive endonucleases. The as-proposed strategy has also been successfully applied to analyze Dam spiked in human serum samples and to assess the inhibitory effects of antibiotics on Dam activity. More importantly, this label-free and truly immobilization-free PEC biosensing strategy shows additional merits of simplicity and satisfactory repeatability, due to the elimination of both labelling and immobilization procedures, making it a promising candidate for the application in highly sensitive, facile and reliable bioanalysis and drug screening., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A biosensor with all the advantages of ultra-high sensitivity, easy operation, straightforward signal output and universal applicability is introduced. The biosensor was demonstrated to work well in the detection of polynucleotide kinase and DAM methyltransferase, thus providing a powerful tool for clinical diagnosis, drug screening and disease therapeutic assay.

DNA methylation is catalyzed by DNA methyltransferase (MTase) and concerned with many biological processes including pathogenesis of various human diseases. The monitoring of MTase activity is thus of great significance in disease diagnosis and drug screening. Herein, we developed a facile way to synthesize biocompatible invertase enzyme modified metal-organic framework (Invertase/MOF) materials, and explored its application in constructing a dual-response Dam MTase sensor for the first time. By using them as signal probes, in which high density of metal sites could be electrochemically detected and invertase could hydrolyze sucrose into glucose for generation of glucometer signal output, dual-response for accurate detection of Dam MTase was realized. In the presence of Dam MTase, the methylation of hairpin probe 1 (HP1) occurred and thus caused the cleavage of HP1 assisted by a restriction endonuclease (DpnI) to produce the binding sequences. The binding sequences then hybridized with the electrode-assembled HP2 to expose their sticky termini which sequentially hybridized with the Invertase/MOFs-tethered capture probes. Finally, the electrodes were incubated with a sucrose solution, followed by the separate electrochemical and glucometer detection. The present assay brought good performance which could detect Dam MTase activity as low as 0.001 U mL -1 with wide linear range and good selectivity against other cytosine MTase (M.SssI MTase). Moreover, it also owns ability to be potentially applied for the inhibitors screening by utilization of 5-fluorouracil as an inhibitor model. The results imply that our proposed method provides a convenient platform for early cancer diagnosis and therapeutic applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED-induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation-sensitive endonuclease DpnⅡ which could recognize the same specific site 5'-GATC-3' with Dam MTase in double-stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME-LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5-20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME-LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA methyltransferase (MTase) is an important epigenetic modification enzyme responsible for DNA methylation, and the dysregulation of DNA MTase activity is associated with various diseases in humans. Herein, we take advantage of the DNA lesion repair mechanism in vivo to develop a new fluorescence approach for the specific and sensitive detection of DNA methyltransferase (DNA MTase) on the basis of the DNA lesion repair-directed cascade isothermal amplification. Due to the high amplification efficiency of the uracil repair-mediated exponential isothermal amplification reaction (EXPAR), the efficient cleavage of endonuclease IV (Endo IV)-induced cyclic catalysis, and the low background signal caused by single uracil repair-mediated inhibition of nonspecific amplification, this approach exhibits high sensitivity with a detection limit of 0.014 U mL -1 for pure Dam MTase and 0.61 × 10 -6 mg mL -1 for Dam MTase in E. coli cells and it can be further applied for the screening of DNA MTase inhibitors. More importantly, this approach can be applied to detect other DNA MTases by designing appropriate substrates, showing great potential in biomedical research and clinical diagnosis.

DNA methyltransferase (MTase) is related to transcriptional repressor activity in biological functions. It is an essential for cancer diagnosis and therapeutics to detect DNA MTase activity sensitively. Here, a fluorescent system based on polymerase amplification has been developed to detect DNA adenine MTase (Dam) activity sensitively. The amplification is triggered by the probe DNA regions a, which are the primes of a polymerase-induced replicated reaction. They come from methylation and a digestion reaction of DNA S1-S1, including a 5'-GATC-3' sequence recognized by Dam MTase and methylation sensitive restriction endonuclease Dpn I. The intensities of fluorescence are dependent on the Dam MTase activity. The method shows fine sensitivity with a detection limit of 3.2 × 10 -4 U mL -1 and specificity for Dam MTase. In human serum samples, the method has been successfully applied, and it has also been used to screen the inhibitors, which means that the developed method can be a powerful and potential tool for drug development and clinical diagnosis in the future.

DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes. However, conventional assays proposed for DNA methyltransferase activity are laborious and discontinuous. We have proposed a novel method for real-time monitoring of the activity and kinetics of Escherichia coli DNA adenine methyltransferase (Dam) using nanopore technique coupled with enzyme-linkage reactions. A double-stranded DNA probe AB having a recognition sequence 5'-GATC-3' for both Dam and MboI restriction endonuclease was prepared. Dam catalyzed the methylation of substrate probe AB, which blocked the cleavage reaction of MboI, while the absence of Dam resulted in cleavage of nonmethylated probe AB into four ssDNA fragments by MboI. When tested with nanopore, double-stranded methylated probe AB generated long-lived events, distinguished clearly from MboI-cleavage-mediated ssDNA fragments that generated only spikelike events. The proposed method has a detection limit of 0.03 U/mL for Dam in a short assay time of about 150 min. This sensing system is easy to perform, simple to design and circumvents the use of radioactive substances, resulting in efficient detection of the activity of Dam even in complex matrixes like human serum sample. Furthermore, it has the potential to screen Dam-targeted inhibitor drugs which may assist in the discovery of new anticancer medicines. This method is general and could be extended easily for monitoring activity of a wide variety of methyltransferases by coupling with their corresponding methylation-sensitive endonucleases.

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg 2+ -dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg 2+ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10 -4  U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, a highly sensitive electrochemical DNA methyltransferase (MTase) activity assay was fabricated with DNA-gold nanoparticles (Au NPs) network as signal amplification unit and an easy assembly method by the linkage of benzenedithiol bridge. By two complementary AuNPs modified single-stranded DNA, DNA-gold nanoparticles network was self-assembled. With the linkage of benzenedithiol bridge, the DNA network structure was immobilized on the surface of gold electrode through the covalent Au-S bond. In the presence of Dam MTase, the special sites of DNA-AuNPs network were methylated and could not be digested by restriction endonuclease Mbo I. Thus the loaded electrochemical indicator Methylene blue (MB) was MB molecules still remained on the DNA-Au NPs network. The electrochemical response depended on the methylated degree, which could be used to detect MTase activity. By the differential pulse voltammetry (DPV), it was demonstrated that a linear relationship between the DPV response and logarithm of Dam concentration ranged from 0.075 to 30 U/mL, achieving a low detection limit of 0.02 U/mL. The use of benzenedithiol avoided the direct incubation of the solid electrode with the capture DNA probe under complex and harsh conditions. Therefore the immobilization of DNA-AuNPs network was easy to be carried out, which is favorable for the specially high stability and reproducibility of the electrochemical biosensor., (Copyright © 2016. Published by Elsevier B.V.)

Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10(-5)U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the target adenine or cytosine, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Herein, we developed a novel electrochemiluminescence biosensor to quantify DNA adenine methylation (Dam) methyltransferase (MTase) employing signal amplification of GO/AgNPs/luminol composites to enhance the assay sensitivity. The method was developed by designing a capture probe DNA, which was immobilized on gold electrode surface, to hybridize with azide complementary DNA to form the azide-terminated dsDNA. Then, alkynyl functionalized GO/AgNPs/luminol composites as the signal probe were immobilized to azide-terminated dsDNA modified electrode via click chemistry, resulting in a high electrochemiluminescence (ECL) signal. Once the DNA hybrid was methylated (under catalysis of Dam MTase) and further cleaved by Dpn I endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5'-G-Am-T-C-3'), GO/AgNPs/luminol composites release from the electrode surface to the solution, leading to significant reduction of the ECL signal. The change of the ECL intensity is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination. This novel strategy can be further used as a universal method for other transferase determination by designing various transferase-specific DNA sequences. In addition, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit of 0.25 U mL(-1), which might make this method a good candidate for monitoring DNA methylation in the future.

The analysis of DNA methylation and MTase (methyltransferase) activity is important in epigenetic study. We have developed a novel strategy for sensitive analysis of MTase activity based on a hairpin shaped DNAzyme; 8-17 DNAzyme amplicon has been adopted and found to be very effective in such analysis.

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Health schemes are promoting application of molecular diagnosis and screening in peripheral diagnostics labs. Intragenic restriction fragment length polymorphisms in the intron 18 (BclI), intron 19 (HindIII), and intron 22 (XbaI) of the Factor VIII gene were studied in 100 patients with hemophilia A and their relatives at risk from different regions of north India. For Bcl I, HindIII, and XbaI, the positive allele frequency was 0.57, 0.38, and 0.43, respectively, and heterozygosity was 0.54, 0.49, 0.41, respectively, whereas the heterozygosity in terms of informativity of the above markers was 53% for BclI, 44% for HindIII, and 34% for XbaI. Combined informativity of these markers was 77%. Review of Indian and world literature shows a marked variation in the informativity of polymorphic sites. Screening for carriers forms the baseline for prevention of hemophilia A. Polymerase chain reaction-restriction fragment length polymorphism is a low-cost procedure, efficient in the north Indian population.

Background and Objective: The plasminogen activating system is a protease/inhibitor system central to extracellular matrix remodeling with a suggested role in periodontal disease pathology. A few studies have reported polymorphisms in the genes of plasminogen activator inhibitors to be associated with periodontal disease severity. Two gene polymorphisms - a BamHI restriction fragment length polymorphism in the urokinase plasminogen activator gene (uPA) and a HindIII restriction fragment length polymorphism in the plasminogen activator inhibitor type 1 gene (PAI-1) - have been associated with conditions having a vascular component, and our objective was to assess the association of these gene polymorphisms with alveolar bone loss in chronic periodontal disease of adults., Material and Methods: Genotype was determined by polymerase chain reaction amplification of whole blood, pertinent histories were obtained by interview, and alveolar bone loss was assessed from current radiographs., Results: In 77 elderly patients with a normal distribution of alveolar bone loss, we demonstrated a significant association between levels of alveolar bone loss and these polymorphisms in the uPA and PAI-1 genes. Controlling for the contributions of smoking or diabetes to periodontal bone loss, estimated odds ratios for predicting lower levels of alveolar bone loss, associated with a greater degree of periodontal health, were strongest when defined by the concurrent presence of a homozygous urokinase plasminogen activator genotype and the nuclease-sensitive plasminogen activator inhibitor type 1 (HindIII) allele (odds ratio = 2.6; 95% confidence interval: 5.8-1.3)., Conclusion: The urokinase plasminogen activator (BamHI) and plasminogen activator inhibitor type 1 (HindIII) genotypes may serve as useful markers for heritability of bone loss associated with periodontal disease.

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.

We show here that the in vivo methylation-based tagging technique DamID (DNA adenine methyltransferase identification) can be used for studies of DNA-protein interactions or chromatin profiling in plants. We have demonstrated the feasibility, reproducibility and sensitivity of the method in Arabidopsis thaliana, using the well-known yeast GAL4 transcription factor, for which DNA-binding sites (UAS(G)) were introduced into the plant genome. We monitored the methylation resulting from the activity of DNA adenine methyltransferase fused to the protein of interest, by combining digestion with methylation-sensitive restriction enzymes and quantitative PCR. We then used DamID to identify genomic targets of LHP1, a protein mostly associated with euchromatin. We showed that LHP1 was targeted to the promoter and transcribed regions of four genes: AG, AP3, FT and PI. Our data also demonstrate that LHP1, like its animal homologues, has a high binding affinity for A/T-rich regions, binding particularly strongly to the large regulatory introns of AG and PI. We identified three major characteristics of LHP1 binding, highlighting the similarities between plant LHP1 and animal HP1 proteins.

Restriction endonucleases (RENs) were detected in 650 microbial strains isolated from water columns and bottom sediments of deep rift lakes, Baikal (Russia) and Nyasa (Southeastern Africa). They enzymes included unique (Fan I, Aca I, and Sse 91) and very rare (Bsi I, and Cci N I) species not typical of aquatic ecosystems. Water columns, deep cores, and bottom sediments of pure areas of the lakes contained no microorganisms with new RENs. Thus, inshore areas of Lake Baikal exposed to anthropogenic influence may contain mutant bacterial strains expressing RENs that have not been described previously.

The plasma cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport (RCT) by mediating the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new polymorphism located at position -971 in the human CETP gene promoter, which corresponds to a G/A substitution at a potential AvaI restriction site. The relationship between the -971G/A polymorphism, plasma lipid parameters and plasma CETP concentration was evaluated in the Etude Cas-Témoins de l'Infarctus du Myocarde (control-myocardial infarction cases) cohort, and revealed that the -971G/A polymorphism (A allele frequency: 0.491) was significantly associated with both plasma high-density lipoprotein cholesterol (HDL-C) levels and CETP concentration (P=0.006 and 0.009, respectively). Subjects with genotype -971GG displayed both low HDL-C levels and high plasma CETP concentration, while genotype -971AA subjects displayed the inverse relationship. Evaluation of potential interactions between the -971G/A and the -629C/A or TaqIB polymorphisms demonstrated that the -971G/A polymorphism interacts significantly with the functional -629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels (P=0.0014 and 0.012, respectively), but does not affect plasma CETP concentration. These results clearly suggest that the interaction between the 971G/A polymorphism and either the -629C/A or the TaqIB polymorphism on plasma CETP concentration is different than that implicated in HDL-C levels. Transient transfection of HepG2 cells revealed that the -971G/A polymorphism did not modulate transcriptional activity of the human CETP gene promoter. The -971G/A promoter polymorphism therefore constitutes a non-functional marker. Furthermore, the observed effects of the -971G/A polymorphism on both plasma CETP concentration and HDL-C levels are due to functional variants in linkage disequilibrium with it. Our findings strongly suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gene and both plasma HDL-C and CETP concentrations.

The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.