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PubMed1979

Organization, replication and modification of the human genome: temporal order of synthesis and methylation of two classes of HeLa nDNA separated in Ag+--Cs2-SO4 gradients.

During the HeLa S-phase, DNA was methylated, at 1-hr intervals in isolated nuclei and fractionated in Ag+-Cs2SO4 gradients providing a heavy GC-rich peak and a main light AT-rich peak. Both size and specific methylation of these peaks changed during the nDNA duplicative phase. Replication of the heavy GC-rich nDNA fraction, which contains genes for ribosomal RNA, occurred in early S; in contrast, replication of the main AT-rich nDNA fraction was maximal in late S. Concomitantly, specific methylation of the GC-rich nDNA was maximal in the first part of S, while that of the AT-rich nDNA was maximal in the second part of S. This suggested that genes are replicated and methylated with order during the S-phase.

Cell Cycle↗

PubMed1981

Selective and potent beta 2-adrenoceptor agents within the tetrahydroisoquinoline class: effect of methyl substitution at the benzylic carbon of the 1-(3,4,5-trimethoxybenzyl) group of trimetoquinol.

A systematic series of methyl (2 and 3) and dimethyl (4) analogues of trimetoquinol (1) were synthesized and evaluated for their beta 1 (atria) and beta 2 (trachea) and adrenoceptor activities. Structural assignments for the erythro (2) and the threo (3) diastereoisomers of 1-(3,4,5-trimethoxy-alpha-methylbenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were based on NMR spectra of the 6,7-dibenzyl precursors 15 and 16, respectively, and on the synthetic derivatives of cis- and trans-13-methyl-2,3-bis(benzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine (18 and 17). The rank order of beta 2-agonist activity for these compounds was 3 greater than 1 greater than 2 greater than 4. The rank order of activity as beta 1 agonists on the guinea pig atria is 1 greater than 3 greater than 2, and 4 was inactive. The methylated analogues show selectivity for beta 2 receptors in our preliminary pharmacological studies. The threo isomer 3 is the most potent and selective beta 2 stimulant reported to date in the tetrahydroisoquinoline class.

Adrenergic beta-Agonists↗

PubMed1986

Developmental and tissue-specific regulation of the Q10 class I gene by DNA methylation.

The H-2 class I genes encode cell-surface glycoproteins that play a critical role in the immune presentation of aberrant cells. The Q10 class I gene, however, encodes a secreted glycoprotein that is highly homologous to the membrane-bound molecules. While the H-2 genes are activated in all tissue types, the expression of the Q10 gene is restricted to only the liver. Analysis of DNA from different tissues revealed a unique methylation profile for the Q10 gene in liver. Developmental activation of this gene in newborn mice is also reflected by a coordinated temporal change in DNA methylation. By comparing the methylation profiles between congenic mice, which differed in their levels of expression of the Q10 gene, it is observed that methylation at the 3'-flanking region correlates with expression. Methylations were at both CG and CC sequences. Since treatment of newborns with 5-azacytidine, which led to inhibition of methylation, resulted in the suppression of Q10, we conclude that hypermethylation in the 3'-flanking region is responsible, at least in part if not in full, for the activation of the Q10 gene in the liver.

Animals↗

PubMed1978

Organization, replication and modification of the human genome: differential methylation of two classes of HeLa nuclear DNA separated on Ag+--Cs2SO4 gradients.

HeLa nuclear DNA sediments as a single peak, in neutral CsCl, while it is separated in a heavier and a lighter components, in alkaline Ag+--Cs2SO4. The heavy fraction, on the average, represents about 20% of the total DNA. CsCl analytical ultracentrifugation shows that heavy DNA bands at 1.715 g/cm3 and contains 53% GC (10% of the total GC), whereas light DNA bands at 1.703 g/cm3 and contains 40% GC (32% of the total GC). Coherently, Tm values in 0.1 x SSC are 82.5 degrees C, for heavy DNA, and 72.5 degrees C, for light DNA. After treatment with [3H-methyl-S-adenosyl-L-methionine in isolated nuclei, the concentration of labelled 5-methylcytosine was found to be highest in the more dense regions of the heavy peak and in the less dense regions of the light peak. Exposure to ultrasound modifies the quantitative relationship of the two peaks and improves the separation of supermethylated AT- and GC-rich DNAs. Four possible triplets as sites for DNA-methylase recognition are discussed.

Cell Nucleus↗

PubMed1998

Angelman syndrome: correlations between epilepsy phenotypes and genotypes.

We compared epilepsy phenotypes with genotypes of Angelman syndrome (AS), including chromosome 15q11-13 deletions (class I), uniparental disomy (class II), methylation imprinting abnormalities (class III), and mutation in the UBE3A gene (class IV). Twenty patients were prospectively selected based on clinical cytogenetic and molecular diagnosis of AS. All patients had 6 to 72 hours of closed-circuit television videotaping and digitized electroencephalogrpahic (EEG) telemetry. Patients from all genotypic classes had characteristic EEGs with diffuse bifrontally dominant high-amplitude 1- to 3-Hz notched or triphasic or polyphasic slow waves, or slow and sharp waves. Class I patients had severe intractable epilepsy, most frequently with atypical absences and myoclonias and less frequently with generalized extensor tonic seizures or flexor spasms. Epileptic spasms were recorded in AS patients as old as 41 years. Aged-matched class II, III, and IV patients had either no epilepsy or drug-responsive mild epilepsy with relatively infrequent atypical absences, myoclonias, or atonic seizures. In conclusion, maternally inherited chromosome 15q11-13 deletions produce severe epilepsy. Loss-of-function UBE3A mutations, uniparental disomy, or methylation imprint abnormalities in AS are associated with relatively mild epilepsy. Involvement of other genes in the chromosome 15q11-13 deletion, such as GABRB3, may explain severe epilepsy in AS.

Adolescent↗

PubMed2002

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.

Amino Acid Sequence↗

PubMed1994

De novo methylation of an MHC class I transgene following transformation with human adenoviruses is not correlated with its altered expression.

The biological importance of class I histocompatibility antigens in a large variety of immune mechanisms is widely recognized, and their role in tumor rejection has been proven in several experimental tumor systems. Reduced expression of class I antigens, which is correlated with enhanced tumorigenicity, was shown in these systems to be mainly the result of transcriptional down-regulation. Mouse embryonal fibroblasts expressing H-2 antigens and the product of a miniature swine class I transgene, transformed by adenovirus 12, exhibit low levels of all class I antigens on the cell surface. Half of the cell lines demonstrate a suppressed level of class I mRNAs. Cell lines derived from transformation with the early region of adenovirus 5 express a high level of class I antigens. DNAs from adenovirus-transformed cells are extensively hypermethylated both in the 5' and the coding regions of the transgene compared to DNAs from immortalized cell lines and primary embryonal fibroblasts. Nevertheless, hypermethylation of these sequences is not correlated with mRNA level or cell-surface expression of the transgene product. Treatment of the transformed cells with high concentration of 5-azacytidine (5 Aza-C) induced merely a minor enhancement in the expression of class I mRNAs and class I antigens. Thus, this system is a perfect example of where viral transformation is associated with induced methylation of a class I gene, but hypermethylation does not affect its expression. The role of de novo methylation of genes in this system might be associated with transformation, or generation of mutations in CpG-rich sequences.

Adenoviridae↗

PubMed1985

Variation in restriction fragment length and methylation pattern of rat MHC class I genes.

Rat MHC (RT.1) class I genes were analyzed by performing Southern blot analysis of digests of genomic DNA isolated from three inbred rat strains using a human derived HLA cDNA probe specific for class I genes. A large number of hybridizing restriction fragments indicated that the rat MHC is a large multigene family. Polymorphic class I DNA restriction fragments specific for the different haplotypes were found for three restriction enzymes used, indicating a high degree of restriction fragment length polymorphism of RT.1 class I genes. Analysis of the methylation state of class I RT.1 genes showed that they are hypermethylated to an extent that is haplotype specific. Furthermore, variation in number and size of Hpa II fragments were observed when comparing liver and spleen DNA from two rat strains sharing the same haplotype. This indicates methylation variation between these organs for RT.1 class I genes.

Animals↗

PubMed1995

Discovery of 6,11-ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations, a novel class of N-methyl-D-aspartate antagonists.

6,11-Ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations 8, a novel class of N-methyl-D-aspartate (NMDA) antagonists acting at the phencyclidine site, have been identified. Structure-activity relationship studies around the lead compound 8a led to the identification of 12g (WIN 67870-2), one of the most potent compounds in this series. Compound 12g has a Ki = 1.8 +/- 0.2 nM vs [3H]TCP binding, has 700-fold selectivity for binding to the open state of the NMDA receptor-ionophore, and was devoid of MK-801- and PCP-like behavioral effects in rats. Compound 12g was neuroprotective in cultured mouse cortical neurons and exhibited antiischemic activity in a rat middle cerebral artery occlusion/reperfusion model of focal ischemia.

Animals↗

PubMed1993

Differences between human sperm and somatic cell DNA in CpG methylation within the HLA class I chromosomal region.

PROBLEM: We investigated the possible negative regulatory mechanisms that repress classical human leukocyte antigen (HLA) class I gene expression in human spermatozoa and searched for novel testis-specific coding sequences that might be present in MHC class I chromosomal region. METHOD: We performed a comparative DNA methylation analysis of this genomic region in both purified human spermatozoa and mononuclear blood cells from the same donors, using methylation-sensitive restriction enzymes followed by classical or pulsed field gel electrophoresis and hybridization with HLA class I locus-specific probes. RESULTS: Unmethylated CpG sites were detected in the 3' part of HpaII tiny fragments of the HLA-F and HLA-G genes in spermatozoal DNA. In contrast, no difference was observed in the methylation status of the HLA-B, HLA-C, and HLA-E genes between germ and somatic cells. CpG unmethylation events were also detected in several parts of this chromosomal region (outside the known loci) in spermatozoal DNA. CONCLUSIONS: These results suggest that this genomic region undergoes changes in its DNA methylation pattern during the developmental process. We hypothesize that these dynamic changes have functional importance, including a possible transcriptional activity of nonclassical class I genes and/or as yet undescribed testis-specific coding sequences.

Base Sequence↗

PubMed1990

Methyl vinyl sulphone: a new class of Michael-type genotoxin.

Methyl vinyl sulphone (MVS) is a labile, Michael-reactive chemical, similar in structure to acrylamide (AA). Given that acrylamide is a reference mammalian mutagen and a rodent carcinogen, studies were undertaken to evaluate the potential genotoxicity of MVS. In common with AA, MVS was non-mutagenic to Salmonella but active as an aneugen to cultured mammalian cells. It is concluded that vinyl sulphones should be regarded as representative of a new class of genotoxic chemical whose mode of action is probably primarily dependent upon Michael reactivity to proteins.

Acrylamide↗

PubMed2006

Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline.

The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the kcat of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the Km and little effect on the Kd of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.

Amino Acid Sequence↗

PubMed1977

Effect of preimmunization with native and methylated carrier on class-specific anti-hapten and anti-carrier antibody response.

Effects of priming with bovine serum albumin (BSA) and methylated BSA (MBSA) on the primary anti-2,4-dinitrophenyl (DNP) response were studied in the chicken. A new radioimmunoassay of class-specific antibodies (RIACA) was used to quantitate IgM and IgG antibodies against BSA and DNP. The anti-DNP antibody response after priming with BSA was dose-dependent: large doses of BSA suppressed and the smallest dose augmented it. In contrast, no dose dependence was observed between MBSA and the anti-hapten response. All doses of MBSA enhanced the primary anti-DNP antibody response of both immunoglobulin classes. No corresponding differences in anti-BSA antibodies were observed between BSA- and MBSA-primed groups. This suggests that the anticarrier antibodies as such are not the agent suppressing the anti-hapten response when large doses of native carrier are used for priming. A more probable explanation of this phenomenon is a competition between carrier-specific B cells and hapten-specific B cells for common T cells. A less obvious explanation is the generation of more helper cells or less suppressor cells when priming with MBSA than with BSA.

Animals↗

PubMed2003

Profile of aberrant CpG island methylation along multistep gastric carcinogenesis.

The stomach is one of the organs whose epithelial cells frequently undergo aberrant methylation of CpG islands. To date, several reports on the methylation of various genes in gastric cancer (GC) have been published. However, most of these studies have focused on cancer tissues or a single gene only and gave no information about the methylation status of specific genes in the premalignant stages or the concurrent methylation of other genes in specific lesions. We attempted to investigate methylation of multiple genes in a large sample collection of GC (n = 80), gastric adenoma (GA) (n = 79), intestinal metaplasia (IM) (n = 57), and chronic gastritis (CG) (n = 74). We determined the methylation frequency of 12 genes, including APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, MGMT, p16, p14, RASSF1A, THBS1, and TIMP3, by methylation-specific PCR. Five different classes of methylation behaviors were found: (a). genes methylated in GC only (GSTP1 and RASSF1A), (b). genes showing low methylation frequency (<12%) in CG, IM, and gastric adenoma (GA) but significantly higher methylation frequency in GC (COX-2, hMLH1, p16), (c). a gene with low and similar methylation frequency (8.8-21.3%) in four-step lesions (MGMT), (d). genes with high and similar methylation frequency (53-85%) in four-step lesions (APC and E-cadherin), and (e). genes showing an increasing tendency with or without fluctuation of the methylation frequency along the progression (DAP-kinase, p14, THBS1, and TIMP-3). The average number of methylated genes was 2.7, 3.6, 3.4, and 5.2 per 12 tested genes in CG, IM, GA, and GC, respectively. Aberrant methylation at multiple loci in the same lesions suggests an overall deregulation of the methylation control, which occurs early in multistep gastric carcinogenesis. Our results suggest that tumor-suppressor genes show a gene-type specific methylation profile along the multistep carcinogenesis and that aberrant CpG island methylation tend to accumulate along the multistep carcinogenesis.

Adenoma↗

PubMed2003

Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis.

To date, several reports on methylation of various genes in gastric cancer (GC) have been published. However, most of these studies focused on cancer tissues or a single gene only and gave no information about the methylation status of specific genes in the premalignant stages or about the concurrent methylation of other genes in specific lesions. We attempted to investigate methylation of multiple genes in a large sample collection of GC (n = 80), gastric adenoma (GA) (n = 79), intestinal metaplasia (IM) (n = 57), and chronic gastritis (CG) (n = 74). We determined the methylation frequency of 12 genes, including APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, MGMT, p16, p14, RASSF1A, THBS1, and TIMP3 by methylation-specific PCR. Five different classes of methylation behaviors were found: (1) genes methylated in GC only (GSTP1 and RASSF1A); (2) genes showing low methylation frequency (<12%) in CG, IM, and GA, but significantly higher methylation frequency in GC (COX-2, hMLH1, and p16); (3) a gene with low and similar methylation frequency (8.8-21.3%) in four-step lesions (MGMT); (4) genes with high and similar methylation frequency (53-85%) in four-step lesions (APC and E-cadherin); and (5) genes showing an increasing tendency with or without fluctuation of the methylation frequency along the progression (DAP-kinase, p14, THBS1, and TIMP3). The average number of methylated genes was 2.7, 3.6, 3.4, and 5.2 per 12 tested genes in CG, IM, GA, and GC, respectively. Our results suggest that tumor suppressor genes show a gene type-specific methylation profile and that aberrant CpG island methylation tends to accumulate along the pathway of multistep carcinogenesis.

Adolescent↗

PubMed2001

CpG island methylation in premalignant stages of gastric carcinoma.

There are limited reports on methylation analysis of the premalignant lesions of gastric carcinoma thus far. This is despite the fact that gastric carcinoma is one of the tumors with a high frequency of CpG island hypermethylation. To determine the frequency and timing of hypermethylation during multistep gastric carcinogenesis, non-neoplastic gastric mucosa (n = 118), adenomas (n = 61), and carcinomas (n = 64) were analyzed for their p16, human Mut L homologue 1 (hMLH1), death-associated protein (DAP)-kinase, thromobospondin-1 (THBS1), and tissue inhibitor of metalloproteinase 3 (TIMP-3) methylation status using methylation-specific PCR. Three different classes of methylation behaviors were found in the five tested genes. DAP-kinase was methylated at a similar frequency in all four stages, whereas hMLH1 and p16 were methylated in cancer samples (20.3% and 42.2%, respectively) more frequently than in intestinal metaplasia (6.3% and 2.1%, respectively) or adenomas (9.8% and 11.5%, respectively). However, hMLH1 and p16 were not methylated in chronic gastritis. THBS-1 and TIMP-3 were methylated in all stages but showed a marked increase in hypermethylation frequency from chronic gastritis (10.1% and 14.5%, respectively) to intestinal metaplasia (34.7% and 36.7%, respectively; P < 0.05) and from adenomas (28.3% and 26.7%, respectively) to carcinomas (48.4% and 57.4%, respectively: P < 0.05). The hMLH1, THBS1, and TIMP-3 hypermethylation frequencies were similar in both intestinal metaplasia and adenomas, but the p16 hypermethylation frequency tended to be higher in adenomas (11.5%) than in intestinal metaplasia (2.1%; P = 0.073). The average number of methylated genes was 0.6, 1.1, 1.1, and 2.0 per five genes per sample in chronic gastritis, intestinal metaplasia, adenomas, and carcinomas, respectively. This shows a marked increase in methylated genes from non-metaplastic mucosa to intestinal metaplasia (P = 0.001) as well as from premalignant lesions to carcinomas (P = 0.002). These results suggest that CpG island hypermethylation occur early in multistep gastric carcinogenesis and tend to accumulate along the multistep carcinogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1993

HLA-E is the only class I gene that escapes CpG methylation and is transcriptionally active in the trophoblast-derived human cell line JAR.

Polymorphic as well as HLA-F and -G genes are repressed in the human cell line JAR, derived from a tumor of trophoblast origin. By contrast, the HLA-E gene as well as the non-HLA novel coding-sequence, R1, located 5' to HLA-E, both remain transcriptionally active. We first demonstrated the role of DNA methylation in the repression of class I genes (except HLA-E) in JAR by the use of the 5-Azacytidine demethylating agent. Following treatment, JAR clones reexpressed polymorphic class I transcripts and cell surface alpha chains. Using methylation-sensitive rare cutter enzymes on JAR genomic DNA, followed by classical or pulse field gel electrophoresis and hybridization with HLA locus-specific probes, we found methylated CpG islands in the 5' region of all class I genes, except for HLA-E. These results, establishing an inverse relationship between states of methylation and transcriptional activity within the MHC class I chromosomal region in JAR, and the observations that the HLA-E and R1 genes were ubiquitously expressed, suggest that the HLA-E chromosomal domain might have functional importance including the presence of housekeeping genes.

Azacitidine↗

PubMed1988

DNA damage-inducible transcripts in mammalian cells.

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury.

Acetoxyacetylaminofluorene↗