Cytosine Residues In DNA Research Articles

De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus.

The relationship between co-suppression and DNA methylation was explored in transgenic plants showing inducible co-suppression following infection with a cytoplasmically replicating RNA virus. Induction resulted in a loss of transgene mRNA and resistance to further infection, factors typical of post-transcriptional gene silencing. In infected plants, de novo methylation of the transgene appeared to precede the onset of resistance and only occurred in plants where the outcome was co-suppression. The methylation was limited to sequences homologous to the viral RNA and occurred at both symmetric and non-symmetric sites on the DNA. Although methylation is predicted to occur in mitotic cells, the virus was found not to access the meristem. A diffusible sequence-specific signal may account for the epigenetic changes in those tissues.

The specific features of methionine biosynthesis and metabolism in plants.

Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for de novo synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of S-adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.

DNA METHYLATION IN PLANTS.

Methylation of cytosine residues in DNA provides a mechanism of gene control. There are two classes of methyltransferase in Arabidopsis; one has a carboxy-terminal methyltransferase domain fused to an amino-terminal regulatory domain and is similar to mammalian methyltransferases. The second class apparently lacks an amino-terminal domain and is less well conserved. Methylcytosine can occur at any cytosine residue, but it is likely that clonal transmission of methylation patterns only occurs for cytosines in strand-symmetrical sequences CpG and CpNpG. In plants, as in mammals, DNA methylation has dual roles in defense against invading DNA and transposable elements and in gene regulation. Although originally reported as having no phenotypic consequence, reduced DNA methylation disrupts normal plant development.

Developmental exposure of male germ cells to 5-azacytidine results in abnormal preimplantation development in rats.

Methylation of cytosine residues in mammalian DNA is established during gametogenesis and embryogenesis; it plays an important role in gene regulation and normal embryonic development and has also been implicated in genomic imprinting. In the present study, we evaluated whether paternal administration of 5-azacytidine, a drug that is incorporated into DNA and blocks DNA methylation, could alter male germ cell development and function. A drug that does not block methylation, 6-azacytidine, served as a control. Adult male Sprague-Dawley rats (n = 4-8 per group) were treated i.p., three times per week for 4 and 11 wk, with saline or 2.5 (low dosage) or 5.0 (high dosage) mg/kg of 5-azacytidine and 6-azacytidine. After each of the treatment periods, males were mated to determine effects on fertility and embryo development. Although neither 6-azacytidine nor 4 wk of 5-azacytidine treatment affected male reproductive organ weights or sperm counts, 11 wk of 5-azacytidine resulted in dose-dependent reductions in testis and epididymal weights and sperm counts. Both dosages of 5-azacytidine resulted in significant increases in preimplantation loss, and the high dosage of 5-azacytidine caused a decrease in fertility. Examination of embryos on Day 2 of gestation revealed a striking dose-dependent increase in the average number of abnormal embryos per litter sired by the males treated with 5-azacytidine (saline, 0.33 +/- 0.24; low dosage, 2.64 +/- 0.92; high dosage, 10.09 +/- 0.95). In summary, paternal administration of 5-azacytidine interfered with normal male germ cell development and resulted in alterations in fertilization and early embryo development. We suggest that 5-azacytidine-induced alterations in germ cell DNA methylation patterns may be one of the underlying mechanisms, since similar dosages of the analogue 6-azacytidine had no effect on male reproduction and progeny outcome.

Deamination of single-stranded DNA cytosine residues in aerobic nitric oxide solution at micromolar total NO exposures.

Deamination of cytosine to uracil is a potential source of mutations in DNA. Here we examine the deaminating ability of aerobic nitric oxide (NO) toward single-stranded DNA at very low (micromolar and below) total exposures, using a sensitive genetic method that allows us to study a single deamination event at a specific site in a 7200-nucleotide DNA molecule within a pool of ca. 100,000 other identical DNA molecules. We incubated gapped C141 M13mp2 DNA with the NO-generating compound, Et2N[N(O)NO]Na (DEA/NO), in aerobic buffer for 16 h to ensure complete autoxidation at pH 7.4 and 37 degrees C. After ultrafiltration to remove small molecules, the DNA was transformed into isogenic Escherichia coli cultures that were either deficient (NR9404, ung-) or proficient (MC1061, ung+) in uracil-DNA glycosylase activity. The gapped DNA was constructed such that the target (CCC) codon was contained in a short single-stranded segment of otherwise double-stranded circular DNA, and the incubation was performed in a closed system to prevent loss of NO to the atmosphere before the reaction was complete. An increase in the reversion frequency in the ung- strain was noted between 0 and 1 microM DEA/NO, and the reversion frequency leveled out between 3 and 30 microM. However, 30 microM "spent" DEA/NO (i.e., that which was similarly incubated for 4 h to complete the autoxidation of NO before the DNA was added) did not increase reversion frequency relative to control. Nearly all (42/43) of the mutations identified after 1 microM DEA/NO treatment were C-->T transitions, and reversion frequency in the isogenic ung+ strain was lower than in the ung- strain. The data are consistent with the hypothesis that total NO exposures in the mumol/L range can lead to C-->T mutations via a mechanism most probably involving deamination of DNA cytosine residues.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

The dam and dcm strains of Escherichia coli — a review

The construction of a variety of strains deficient in the methylation of adenine and cytosine residues in DNA by the methyltransferases (MTases) Dam and Dcm has allowed the study of the role of these enzymes in the biology of Escherichia coli. Dam methylation has been shown to play a role in coordinating DNA replication initiation, DNA mismatch repair and the regulation of expression of some genes. The regulation of expression of dam has been found to be complex and influenced by five promoters. A role for Dcm methylation in the cell remains elusive and dcm − cells have no obvious phenotype. dam − and dcm − strains have a range of uses in molecular biology and bacterial genetics, including preparation of DNA for restriction by some restriction endonucleases, for transformation into other bacterial species, nucleotide sequencing and site-directed mutagenesis. A variety of assays are available for rapid detection of both the Dam and Dcm phenotypes. A number of restriction systems in E. coli have been described which recognise foreign DNA methylation, but ignore Dam and Dcm methylation. Here, we describe the most commonly used mutant alleles of dam and dcm and the characteristics of a variety of the strains that carry these genes. A description of several plasmids that carry dam gene constructs is also included.

Selective Hypermethylation of Transcribed Nucleosomal DNA by Sodium Butyrate

Previous studies have shown that treatment of cultured fibroblasts with millimolar concentrations of sodium butyrate results in increased methylation of cytosine residues in DNA. In this study, active nucleosomes were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its constituent bases and subjected to HPLC analysis in order to determine the 5-methylcytosine content. In control cells, the active nucleosomal DNA was hypomethylated (0.97 ± 0.27% 5-methylcytosine) when compared with the inactive DNA fraction (1.61 ± 0.15%). This result was not unexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resulted in increased methylation of the active nucleosomal DNA such that it was comparable to that of the inactive fraction of control cells (1.73 ± 0.02% 5-methylcytosine). A much smaller increase in 5-methylcytosine content was detected in the inactive DNA fraction of sodium butyrate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrate followed by a chase in butyrate-free medium for up to 120 h failed to reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days.

Chemical synthesis of 2'-deoxyoligonucleotides containing 5-fluoro-2'-deoxycytidine.

2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific positions of the nucleotide sequence are tools of great potential in the study of the catalytic mechanism by which DNA cytosine methyltransferases methylate the 5-position of DNA cytosine residues in specific sequence contexts. Chemical synthesis of such oligonucleotides is described. Two alternative approaches have been developed, one of which proceeds via a fully protected phosphoramidite of 5-fluoro-4-methylmercapto-2'-deoxyuridine 2, the other via a fully protected phosphoramidite of 5-fluoro-2'-deoxycytidine 3. Either building block can be used in automated oligonucleotide synthesis applying standard elongation cycles and deprotection procedures exclusively. The methylmercapto function of 2 is replaced by an amino group in the final ammonia treatment used for cleavage from support and base deprotection.

Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A.T----G.C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G.C----A.T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G.C----A.T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A.T----G.C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.

Spontaneous deamination of cytosine and 5-methylcytosine residues in DNA and replacement of 5-methylcytosine residues with cytosine residues

Methylation of cytosine residues at position 5 is the only known genetically programmed modifica- tion of the bases in vertebrate DNA. 5-Methylcy- tosine (mSC) constitutes - 0.7-3% of the bases of all studied normal vertebrate cells or tissues and is found mostly in CpG dinucleotide sequences (Ehrlich and Wang, 1981; Gama-Sosa et

Potential role of anti-oncogenes in ageing

Some observations on cellular senescence are discussed regarding the possibility that the postulated genes, which bring about the DNA synthesis block of senescent fibroblasts, might be similar to those designated as anti-oncogenes or tumour suppressor genes. The latter genes have been defined by tumorogenicity tests of hybrids from fused tumour and normal cells, of which the retinoblastoma (RB) gene is a prototype. Similarities between the two systems are considered, which are consistent with the assumption that senescent growth arrest, like the suppression of tumorogenicity of the hybrids, might be effected by anti-oncogenes via inhibition of cellular oncogenes in their growth signal transduction functions. A link may be provided by the finding of a direct interaction of certain DNA tumour virus oncoproteins with the RB gene product p105-RB, together with observations that the tumour viruses are also able to reinitiate DNA synthesis in growth arrested senescent fibroblasts. The possibility is also discussed that some silent retinoblastoma-like genes might become aberrantly expressed in senescent cells owing to a progressive loss of 5-methyl cytosine residues of DNA.

Hydrolysis of N3-methyl-2′-deoxycytidine: Model compound for reactivity of protonated cytosine residues in DNA

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N 3-methyl-2′-deoxycytidine (m 3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m 3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

Maturation in Larch

The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log(10) age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types.

DNA Methylation and Differentiation

The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin. Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable.

DNA methylation and differentiation.

The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin. Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable.

A portable signal causing faithful DNA methylation de novo in Neurospora crassa.

Methylation of cytosine residues in eukaryotic DNA is common, but poorly understood. Typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified. Neurospora crassa DNA containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct DNA methylation de novo. DNA stripped of its methylation by propagation in Escherichia coli was reintroduced into Neurospora crassa by transformation. The zeta-eta region reproducibly became "properly" methylated whether inserted at its native chromosomal position or at ectopic sites. Adjacent Neurospora and bacterial sequences in the transforming DNA rarely became methylated. A model is presented that accounts for position-independent faithful methylation as observed in the zeta-eta region, as well as position-dependent methylation, as occasionally observed, especially with sequences not native to Neurospora.

Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2'-deoxycytidine resistance.

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

DiFferential Nuclear Protein Binding to 5-Azacytosine-Containing DNA as a Potential Mechanism for 5-Aza-2′-Deoxycytidine Resistance

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

Role of the main cytosine radiolytic product in ionizing radiation-induced mutagenesis.

When 14C cytosine was incorporated in M13 mp10 phage, in the phage DNA both cytosine and thymine residues were labelled. The single stranded phage DNA was isolated and irradiated in a buffer solution using 60Co gamma-rays. After the addition of TCA, the DNA precipitated was filtered. The ratio of the radioactivity of the filtrate to the total increased with increasing radiation dose. The precipitate -was hydrolyzed with acid and the base modification was analyzed by TLC. The modification yield also increased with increasing dose. Chromatograms showed that the main product was urea. In the case of cytosine radiolysis, trans-5, 6-dihydroxy-5, 6-dihydrouracil was found to be the main product before acid treatment; whereas, urea was the main product after acid treatment. This fact suggests that the trans-5, 6-dihydroxy-5, 6-dihydrouracil retained in the irradiated DNA is responsible for the gamma-ray induced C-T transition.