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PubMedDate not supplied

[Determination and analysis of the primary structure of NM.BstSEI operone from Bacillus stearothermophilus SE-589 which produces N.BstSEI site-specific nickase].

Nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment which includes an operone for site-specific NM-system with a gene for BstSEI nickase has been determined. Analysis of the regions adjacent to nickase gene has revealed two genes encoding DNA methyltransferases, which belong to different classes. Three genes which form system operone are separated with short open reading frames (ORFs). Analysis of these ORFs has shown that they encode polypeptides which are homologous to different parts of BstSEI nickase, NatB protein and arginase. A difference in GC-content of the beginning and ending regions of the cloned DNA fragment as well as presence of short ORFs similar to genes for known proteins may indicate that NM.BstSEI system operone has evolved by horizonthal DNA transfer.

Amino Acid Sequence↗
PubMed2006

p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions.

Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low-grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using real-time methylation-specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low- and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had >or= 2 methylated genes, while 17/19 (89%) of normal and low-grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARbeta2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful in a primary screening test for ESCC and its precursor lesions.

Adult↗
PubMed2006

[Methylation pattern of DNA repair genes and microsatellite instability in hepatocelluar carcinoma].

BACKGROUND/AIMS: Epigenetic silencing of DNA repair genes, O6-methylguanine-DNA methyltransferase (MGMT), hMLH1 and hMSH3, by promoter hypermethylation have been observed in various cancers. However, the relationship between hypermethylation of DNA mismatch repair genes and microsatellite instability (MSI) has not been studied in hepatocellular carcinoma (HCC) associated with cirrhosis. METHODS: We investigated the methylation pattern of CpG islands of 3 genes using methylation-specific PCR (MSP) and MSI in 40 patients with paired hepatocellular carcinoma and associated cirrhosis. RESULTS: hMSH3 and MGMT were the most methylated genes in both cirrhosis (70% and 68%, respectively) and HCC (75% and 73%, respectively). The methylation of hMLH1 was rarely found in both cirrhosis (8%) and HCC (5%). Gene promoters methylated in cirrhosis were also methylated in HCC with the exception of 9 cases found to be methylated either in cirrhosis or HCC. Of 40 cases of HCC associated with cirrhosis, three had MSI-positive phenotype in which two were MSI-low and one was MSI-high. One MSI-positive phenotype was present both in cirrhosis and in HCC, while two were only in HCC. There was no significant correlation between aberrant DNA methylation of mismatch repair genes and MSI status in HCC associated with cirrhosis. Immunohistochemical expressions of hMLH1, MGMT, and hMSH3 proteins were present in 16 (40%), 6 (15%), and 11 (28%) of 40 cases of HCC respectively. There was no significant correlaton between the aberrant DNA methylation of mismatch repair genes and clinical characteristics such as histological differentiation, postoperative recurrence and mortality. CONCLUSIONS: The methylation of MGMT and hMSH3 among DNA repair genes are frequent, but those of hMLH1 and MSI is very rare in both cirrhosis and HCC. There is no significant correlation between the methylation of DNA repair genes and clinical characteristics of HCC.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Growth regulation of mouse DNA methyltransferase gene expression.

The steady state level of DNA methyltransferase mRNA is markedly increased as growth-arrested Balb/c 3T3 cells progress into the S phase of the cell cycle. mRNA abundance is reduced to the basal level before termination of DNA synthesis activity. Maintenance DNA methylation activity in nuclear extracts follows a similar pattern with two exceptions. (a) A small peak of DNA methylation activity is detected in early G1 phase. (b) The extinction of DNA methylation activity lags behind the termination of DNA synthesis. Nuclear runon experiments demonstrate that the gene is transcribed in growth-arrested cells, and expression of the gene is post-transcriptionally regulated. We suggest that this mode of regulation of the DNA methyltransferase gene might play an important role in determining and maintaining DNA methylation patterns.

Animals↗
PubMed1989

[DNA-methylase Sau 3A: isolation and various properties].

DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.

Base Sequence↗
PubMed1987

Enzymatic probes for left-handed Z-DNA.

In conclusion, one of the aspects of the DNA polymorphism observed is the formation of Z-DNA under a variety of conditions. Left-handed DNA stretches not only represent alternate structures, but also exert long-range effects due to their influence on superhelical properties on an entire supercoiled DNA as first shown six years ago [49, 50]. The examples given in this review emphasize the site-specificity of enzymes due to structural features rather than sequence itself. In this fashion, the reversible transition from B to Z DNA could modulate site-specific events on many levels of biological regulation. Considering all of the enzymes studied to date (S1, mung bean, BAL31, P1 nucleases, Hha I, BssH II, MHha I, BamH I, EcoR I, RNA polymerase, recl, recA, DNA glycosylase, O6-methylguanine-DNA methyltransferase), only the recl (and possibly the recA) protein seems to recognize and utilize left-handed DNA. A large number of questions related to the biology of Z-DNA are unanswered including: what is the DNA structure (B or Z or other) which is in physical contact with proteins; is Z-DNA recognized by proteins or are junctions the important features; do proteins revert the Z structure to B or to some other right-handed conformation; what other cofactors (perhaps chiral in nature) may be involved; what are the alternate forms of left-handed DNA; does left-handed DNA exist in vivo; what is the biological role(s) of left-handed DNA? The future of this field of investigation will be exciting indeed.

Computer Simulation↗
PubMed1987

Enhancement of the apparent cleavage specificities of restriction endonucleases: applications to megabase mapping of chromosomes.

We have described how DNA methylases may be used to enhance the apparent specificities of restriction endonucleases [9-11, 17, 18] to generate DNA fragments averaging 6000 to 270,000,000 base pairs on random DNA. Taking into account the non-random arrangement of natural DNA, we have predicted the rarity of certain recognition sequences in the genomes of several species.

Base Sequence↗
PubMed1981

[DNA-methylase from Arthrobacter luteus screens DNA from the action of site-specific endonuclease Alu I].

DNA-methylase was isolated from a cell extract of A. luteus and partially purified by chromatography on phosphocellulose. The purified enzyme methylates DNA of phage lambda and plasmids pBr322, thus making them resistant to a subsequent action of endonuclease Alu I. It has been shown that cytosine is the object of methylation within DNA. This modification does not screen DNA from the action of site-specific endonucleases Sal I, Bam HI, EcoR I, EcoR II, Xho I and Xho II. It has been experimentally demonstrated that the isolated methylase is site-specific and identifies in the DNA the nucleotide sequence 5'-AGCT-3', by methylating cytosine in the DNA.

Arthrobacter↗
PubMed1993

[Isolation of site-specific endonucleases and methylases from Bacillus cereus 83].

The site-specific endonuclease R Bce83I and methylase M Bce83I were isolated from Bacillus cereus 83 by three consecutive chromatographies on blue agarose, hydroxyapatite and heparin-Sepharose. R Bce83I recognizes [formula: see text] sequences on the DNA and cleaves the DNA as indicated by the arrows. The endonuclease is stimulated by S-adenosyl-L-methionine and may consequently be referred to type IV restriction enzymes.

Bacillus cereus↗
PubMedDate not supplied

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes.

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false positives in the cloning stimulation assay were less than 5%. This conditions could ensure a good performance of the enzyme preparations for cloning experiments. Finally, we described the use of the radiolabeled [gamma 33P] ATP lambda Hpa II DNA substrate to detect 5'-->3' single stranded DNA dependent exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I.

Bacteriological Techniques↗
PubMed1996

Exon-specific DNA hypomethylation of the p53 gene of rat colon induced by dimethylhydrazine. Modulation by dietary folate.

Folate deficiency enhances colorectal carcinogenesis in dimethylhydrazine-treated rats. Folate is an important mediator of DNA methylation, an epigenetic modification of DNA that is known to be dysregulated in the early stages of colorectal cancer. This study investigated the effect of dimethylhydrazine on DNA methylation of the colonic p53 gene and the modulation of this effect by dietary folate. Sprague-Dawley rats were fed diets containing 0, 2, 8, or 40 mg of folate/kg of diet. Five weeks after diet initiation, dimethylhydrazine was injected weekly for fifteen weeks. Folate-depleted and folate-replete control animals did not receive dimethylhydrazine and were fed the 0- and 8-mg folate diets, respectively. The extent of p53 methylation was determined by a quantitative HpaII-polymerase chain reaction. In exons 6 and 7, significant p53 hypomethylation was observed in all dimethylhydrazine-treated rats relative to controls (P < 0.01), independent of dietary folate. In exon 8, significant p53 hypomethylation was observed only in the dimethylhydrazine-treated folate-depleted rats compared with controls (P = 0.038) and was effectively overcome by increasing levels of dietary folate (P = 0.008). In this model, dimethylhydrazine induces exon-specific p53 hypomethylation. In some exons, this occurs independent of dietary folate, and in others, increasing levels of dietary folate effectively override the induction of hypomethylation in a dose-responsive manner. This may be a mechanism by which increasing levels of dietary folate inhibit colorectal carcinogenesis.

Animals↗
PubMed1997

Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer.

The tumor suppressor gene CDKN2 (p16/MTS1) resides on chromosome 9p21 and encodes a 16 kDa inhibitor of the cyclin-dependent kinases. Inactivation of CDKN2 by homozygous deletion, point mutation, and recently described aberrant methylation in the 5' promoter region may increase progression through the cell cycle in tumors. In this study, we examine the CDKN2 gene for the presence of inactivating alterations in human prostate cancer. Sequence analysis of cell lines revealed no mutation in LNCaP, PC3, and TSU-PR1 and a missense mutation, GAC-->TAC (asp to tyr), in exon 2 of the DU145 cell line at codon 76. No mutations were identified in three primary prostate cancers or in seven lymph node metastases. Loss of heterozygosity (LOH) was analyzed by analysis of microsatellite markers in the vicinity of the CDKN2 gene. LOH was detected in 12 (20%) of 60 primary tumors at one or more loci and in 13 (46%) of 28 metastases. Methylation analysis of the CpG-rich promoter region revealed a dense methylation of CDKN2 in cell lines PC3, PPC1, and TSU-PR1, and this was found to correlate with a lack of mRNA expression by reverse transcription-polymerase chain reaction. A demethylating agent, 5-aza-2'-deoxycytidine, induced reexpression when cells were exposed in vitro. DU145 and LNCaP expressed the CDKN2 transcript and were unmethylated in the promoter region. Three of twenty-four (13%) primary prostate cancers and 1 of 12 metastatic tumors demonstrated promoter methylation. No normal prostate tissues were methylated at the CDKN2 gene promoter. One tumor was found to contain concomitant LOH and promoter methylation indicative of biallelic inactivation. A comprehensive analysis of CDKN2 in prostate cancer reveals that point mutations are infrequent, but gene deletion and methylation combine to inactivate CDKN2 in a subset of tumors. Moreover, alterations in this gene may represent a late event in prostate cancer progression.

Aged↗
PubMedDate not supplied

Examination of the DNA methylation properties in nontumorigenic and tumorigenic breast epithelial cell lines.

Molecular changes in the progressive state of tumorigenesis often include altered patterns of DNA methylation. Utilizing a series of breast epithelial cell lines, the overall 5-methylcytosine content in genomic DNA demonstrated an overall decrease when comparing two malignant cell lines (MCF-7 and T47D) with a mortal cell line (MCF 1 2M) and several derivative cell lines of the immortalized MCF10 cultures (MCF10A,-2A, -5A, A1neoT2, and 139B6). Further investigation on the methylation status of these cells lines indicated no difference in DNA methyltransferase activity, both at a protein and mRNA levels, in the nontumorigenic cell lines examined while activity was 3-10 fold higher in the tumorigenic lines (MCF7, T47D, SkBr3, MB-MDA-231, -468). Examination of the CpG island in the 5' promoter region of the estrogen receptor gene indicates that this region is unmethylated in the mortal and immortal nontumorigenic lines as well as the tumorigenic lines examined, with the exception of the estrogen receptor negative breast cell line MB-MDA-468 which appears to be partially methylated at this site. These results indicate methylation of this CpG island does not account for the inactivation of the estrogen receptor gene in immortalized nontumorigenic breast cells, suggesting another mechanism of transcriptional inactivation of ER in this environment.

Breast↗
PubMed1981

Alternative conformations of DNA modified by N-2-acetylaminofluorene.

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the "base displacement model" with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S1 digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S1 digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC).(dG-dC). Modification of his copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. Poly(dG-dC).poly(dG-dC) which does not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modifications. Differences in conformation were suggested by single-strand specific nuclease S1 digestion and reactivity with anticytidine antibodies. Highly modified poly(GS-dC).poly(DG-dC) was almost completely resistant to nuclease S1 hydrolysis, while, modified DNa and poly(dG).poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.

2-Acetylaminofluorene↗