Cytosine Residues In DNA Research Articles

A method for evaluating correlation times for tumbling and internal motion in macromolecules using cross-relaxation rate constants from proton NMR spectra

A method of estimating the correlation times and extent of internal motion of macromolecules using 1H NMR is proposed. The method relies on measuring the cross-relaxation rate constant between resolved, identified protons separated by a fixed distance, for example 2, 3 protons of tyrosine residues or 4, 5 protons of tryptophan residues in proteins, and the 5, 6 protons of cytosine residues in DNA. For a rigid body, the cross-relaxation rate constant yields directly an estimate of the tumbling time. Deviation of its dependence on viscosity and temperature from expectations for a rigid body allows one to estimate the degree to which internal motions contribute to the relaxation. The method is illustrated for Ribonuclease A and a 20 base pair fragment of DNA corresponding to the trp operator of Escherichia coli. The calculated correlation time of RNAse A is about 8 ns at 298 K, in good agreement with expectations from hydrodynamic measurements. Tyrosine 25 has significant internal motion, characterized by an apparent amplitude of 50–60°, a correlation time of about 5 ns, and low activation energy. The correlation time of the fragment of DNA is about 6.4 ns at 298 K, in agreement with expectations for a rigid rod. The apparent activation energy was 3.8 kcal/mol, close to the value for the dependence of the viscosity of D 2O on temperature. Further, the same result was obtained regardless of the position of the base in the sequence, indicating that bending motions are of small amplitude on the nanosecond time scale for short fragments of DNA.

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.

5-azacytidine potentiates initiation induced by carcinogens in rat liver.

To test the validity of the hypothesis that hypomethylation of DNA plays an important role in the initiation of carcinogenic process, 5-azacytidine (5-AzC) (10 mg/kg), an inhibitor of DNA methylation, was given to rats during the phase of repair synthesis induced by the three carcinogens, benzo[a]-pyrene (200 mg/kg), N-methyl-N-nitrosourea (60 mg/kg) and 1,2-dimethylhydrazine (1,2-DMH) (100 mg/kg). The initiated hepatocytes in the liver were assayed as the gamma-glutamyltransferase (gamma-GT) positive foci formed following a 2-week selection regimen consisting of dietary 0.02% 2-acetylaminofluorene coupled with a necrogenic dose of CCl4. The results obtained indicate that with all three carcinogens, administration of 5-AzC during repair synthesis increased the incidence of initiated hepatocytes, for example 10-20 foci/cm2 in 5-AzC and carcinogen-treated rats compared with 3-5 foci/cm2 in rats treated with carcinogen only. Administration of [3H]-5-azadeoxycytidine during the repair synthesis induced by 1,2-DMH further showed that 0.019 mol % of cytosine residues in DNA were substituted by the analogue, indicating that incorporation of 5-AzC occurs during repair synthesis. In the absence of the carcinogen, 5-AzC given after a two thirds partial hepatectomy, when its incorporation should be maximum, failed to induce any gamma-GT positive foci. The results suggest that hypomethylation of DNA per se may not be sufficient for initiation. Perhaps two events might be necessary for initiation, the first caused by the carcinogen and a second involving hypomethylation of DNA.

Detection and measurement by high-performance liquid chromatography of malondialdehyde crosslinks in DNA

Malondialdehyde, which is generated by lipid peroxidation, can form DNA-protein and/or interstrand DNA crosslinks. The biological consequences of inaccurate repair of these crosslinks may be severe. The expected levels of crosslinking of DNA in vivo are low, and an extremely sensitive method must be used for their detection and measurement. Because both types of crosslinks may contain cytosine, the cytosine residues of DNA were labeled in vitro with 125I. The iodinated DNA was treated with Penicillium nuclease P 1 at pH 6 and with alkaline phosphatase at pH 9, and the nucleosidic compounds were analyzed by high-performance liquid chromatography. The optimum conditions for measurement of the crosslinks on Ultrasphere ODS or Zorbax ODS columns were 50 m m ammonium phosphate buffer, pH 7, that contained 2% methanol and 5 m m tetra- t-butylammonium phosphate. Both DNA-protein and interstrand DNA crosslinks were measurable simultaneously. The method was quantitative, reproducible, and able to detect crosslinked adducts at subpicomole levels, so that as few as two crosslinks per 10 6 base pairs were detectable.

The role of DNA methylation in virus replication: Inhibition of frog virus 3 replication by 5-azacytidine

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated ( D. Willis and A. Granoff, 1980, Virology 107, 250–257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 μM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Inhibition of DNA methyltransferases in vitro by benzo[a]pyrene diol epoxide-modified substrates.

Covalent adducts formed from the ultimate carcinogen 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene inhibit the enzyme-catalyzed transfer of methyl groups from S-adenosylmethionine to cytosine residues in DNA. Two DNA methyltransferase enzymes, isolated from the bacterium Haemophilus and mouse spleen nuclei, were tested for their ability to methylate carcinogen-modified substrates in vitro. These model enzymes possess the known methylation activities found in mammalian cells, de novo, and maintenance methylation of CpG-containing nucleotide sequences. The in vitro alkylation of DNA substrates by the carcinogen effectively decreases the methyltransferase reaction of both enzymes in a manner that is directly dependent upon the level of covalent modification of the DNA. Inhibition of de novo methylation activity can be detected at very low levels of carcinogen modification, 1 hydrocarbon residue per 20,000-40,000 nucleotides. Adduct levels in this range are capable of initiating transformation. Both enzymes are inactivated by direct reaction with the carcinogen in the absence of DNA. We also find that carcinogen adducts are capable of inhibiting DNA methylation at CpG sites removed from the primary lesion. These results support the proposal that carcinogen-induced DNA damage can cause alterations in methylation patterns that may eventually lead to heritable changes in gene expression.

Chemical carcinogen-mediated decreases in DNA 5-methylcytosine content of BALB/3T3 cells.

The genomic level of DNA cytosine methylation was significantly diminished in dividing BALB/3T3 A31 CL1-13 cells treated with several aromatic hydrocarbon carcinogens. Benzo[a]pyrene-induced decreases in DNA 5-methylcytosine levels were concentration dependent over the range of 0.1 to 1.0 microgram/ml when determined at the end of the 16 h treatment period. The enzymatic methylation of DNA cytosine residues was the most sensitive to inhibition 48 h after treatment as concentrations of benzo[a]pyrene as low as 0.033 micrograms/ml initiated significant reductions in 5-methylcytosine levels. This inhibition of DNA cytosine methylation may be mediated by alkylation of the DNA. Treatment of hemimethylated DNAs with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene (anti-BPDE), (+/-)-r,7-t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE), and (+/-)-benzo[a]pyrene-4,5-epoxide inhibited the transfer of methyl moieties from [3H]S-adenosylmethionine to cytosine residues in the undermethylated strand in the presence of mouse spleen methyltransferase activity. The syn isomer of BPDE was the most potent in this action while the parent compound, benzo[a]pyrene, did not significantly decrease the methyl accepting abilities of treated DNAs. All chemical carcinogens that were tested and are known to transform BALB/3T3 cells initiated significant reductions in 5-methylcytosine formation by 48 h post-treatment. However, concentrations of some hydrocarbons which do not transform these cells 5- to 50-fold above effective concentrations of the transforming carcinogens also provided significant reductions in DNA cytosine methylation. Thus the inhibition of DNA methylation may be an important step in the initiation of oncogenic transformation of BALB/3T3 cells, but decreases in DNA 5-methylcytosine levels alone cannot account for the onset of this multi-step process.

DNA modification, differentiation, and transformation.

Substantial evidence has accumulated over the last 5 years that the methylation of cytosine residues in vertebrate DNA is implicated in the control of gene expression. We have used analogs of cytidine, modified in the 5 position, as specific inhibitors of DNA methylation to probe the relationship between this process and cellular differentiation. 5-Azacytidine effected marked changes in the differentiated state of cultured cells and induced the formation of biochemically differentiated muscle, fat, and chondrocytes from mouse fibroblast cell lines. Since the analog is a powerful inhibitor of DNA methylation, we suggest that this inhibition is causally related to the mechanism of phenotypic conversion. DNA extracted from cells treated with 5-azacytidine was hemimethylated and was used as an efficient acceptor of methyl groups in an in vitro reaction in the presence of eukaryotic methylases. In vitro methylation was inhibited if the substrate DNA was preincubated with a diverse range of chemical carcinogens including benzo(a)pyrene diolepoxide. Thus, chemical carcinogens may induce changes in gene expression by alteration of cellular methylation patterns. Recent experiments have also demonstrated that freshly explanted diploid fibroblasts from mice, hamsters, and humans lose substantial quantities of 5-methylcytosine during cell division and aging in culture. Taken together, these experiments suggest that the genomic distribution of 5-methylcytosine might have importance in normal differentiation and also in the aberrant gene expression found in cancer and senescence in culture.

Role of de novo DNA methylation in the glucocorticoid resistance of a T-lymphoid cell line.

A correlation has been shown between changes in the methylation pattern of cytosine residues in DNA and the expression of specific genes in differentiated tissues. The pattern of DNA methylation is conserved, through cell division, by a maintenance methylase but the mechanism by which a given pattern of methylation is established is unknown. De novo methylation of foreign DNA molecules has been shown to occur in several systems, and may serve as a signal to arrest gene expression. Conversely, treatment of cultured cell lines with 5-azacytidine results in DNA hypomethylation and leads to transcriptional activation of previously unexpressed genes. The results described here demonstrate spontaneous de novo methylation of DNA in a T-lymphoid cell line previously treated with 5-azacytidine to generate glucocorticoid sensitivity. This de novo methylation is accompanied by the acquisition of the glucocorticoid-resistant phenotype.

Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli.

Studies of trpA reversions revealed that G:C leads to A:T transitions were stimulated about 30-fold in E. coli ung mutants, whereas other base substitutions were not affected. A dUTPase (dut) mutation, which increases the incorporation of uracil into DNA in place of thymine, had no significant effect on the rate of G:C leads to A:T transitions. The results support the proposal that the glycosylase functions to reduce the mutation rate in wild-type cells by acting in the repair of DNA cytosine residues that have undergone spontaneous deamination to uracil. Further support was provided by the finding that when lambda bacteriophages were treated with bisulfite, an agent known to produce cytosine deamination, the frequency of clear-plaque mutants was increased an additional 20-fold by growth on an ung host. Bisulfite-induced mutations of the cellular chromosome, however, were about equal in ung+ and ung strains; it was found that during the treatment of ung+ cells with bisulfite, the glycosylase was inactivated.

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA

5-Methylcytosine residues in DNA underwent deamination at high temperatures. Furthemore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [ 14C]5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [ 14C]5-methylcytosine residues. Upon incubation at 95°C in a physiological buffer or at 60°C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those of cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.

Enzymatic conversion of deoxycytidine 5′-monophosphate to 5-methyldeoxycytidine 5′-triphosphate

An enzymatic method has been developed to synthesize 5-methyldeoxycytidine triphosphate (m 5dCTP) starting with dCMP and formaldehyde as substrates. Methylation is catalyzed by a dCMP methyltransferase in the presence of tetrahydrofolic acid. The resulting m 5dCMP is converted to m 5dCTP by a kinase preparation and an ATP-regenerating system. The methyltransferase and kinase preparations were isolated from extracts of bacteriophage XP-12-infected Xanthomonas oryzae by a single-step chromatography on DEAE-cellulose. About 10% of the input radioactivity from 14C- or 3H-labeled formaldehyde was recovered in m 5dCTP under our reaction conditions. Using this enzymatically synthesized m 5dCTP, up to 60% of the cytosine residues in DNA was replaced by 5-methylcytosine residues in a nick translation reaction catalyzed by Escherichia coli DNA polymerase I.

Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine.

Three cytidine analogs containing modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2'-deoxycytidine and pseudoisocytidine) induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP; pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X chromosome retained in a mouse-human somatic cell hybrid. Between 0.1% and 8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but two other analogs, 5-fluoro-2'-deoxycrytidine and 5.6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X chromosome present in the hybrid, was found to be late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2'-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because these analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.

Extensive methylation of chloroplast DNA by a nuclear gene mutation does not affect chloroplast gene transmission in chlamydomonas

Based on analysis by high pressure liquid chromatography, greater than 35% of the cytosine residues in chloroplast DNA of vegetative cells were found to be methylated constitutively in the nuclear gene mutation ( me-1) of Chlamydomonas reinhardtii, which has an otherwise wild-type phenotype. Digestion of chloroplast DNA from vegetative cells and gametes of this mutant with restriction endonucleases Hpa II and Msp I reveals that in the 5′CCGG3′ sequence, CpG is methylated extensively, whereas CpC is only methylated occasionally. Hae III (5′GGCC3′) digestion of the mutant chloroplast DNA also shows extensive methylation of the GpC sequence. In contrast to the results of Sager and colleagues, which show a correlation between methylation of chloroplast DNA and transmission of chloroplast genes in crosses, our results with crosses of the me-1 mutant suggest that extensive chloroplast DNA methylation may be insufficient to account for the pattern of inheritance of chloroplast genes in Chlamydomonas.

The inheritance of methylation patterns in vertebrates

The genomic DNAs of most organisms contain modified bases. In vertebrates, 5-methyl-cytosine (“C) is the only modified base; it results from enzymatic transfer of the methyl group of S-adenosyl-methionine to cytosine residues in DNA. Most but not all “C occurs in the dinucleotide 5’-CpG, and in mammals and birds approximately 50-70% of all such dinucleotides are modified. Considerable interest in DNA methylation has been created by increasing evidence linking methylation patterns to patterns of gene expression; this subject has been recently reviewed elsewhere (Razin and Riggs, Science 270, 604-610, 1980). The actual distribution of “C within specific genes can be probed with the use of bacterial restriction endonucleases, such as Hpa II and Hha I, that will not cleave recognition sequences containing “CpG. Tissue-specific differences in methylation patterns have been noted (Waalwijk and Flavell. NAR 5, 46314641, 1978; Mandel and Chambon, NAR 7, 20812103, 1979; McGhee and Ginder, Nature 280, 419420, 1979; van der Ploeg and Flavell, Cell 19, 947958, 1980) and strong correlations exist between methylation and transcriptional inactivity of integrated viral genomes (Desrosiers et al., PNAS 76, 38393843, 1979; Cohen, Cell 19, 653-662, 1980; Sutter and Doerfler, PNAS 77, 253-256, 1980). Experiments with DNA-mediated gene transfer have suggested a causal link between methylation and inhibition of gene expression (Pollack et al., PNAS 77, 6463-6467, 1980; Wigler et al., Cell 24, 33-40, 1981). Recent experiments involving oocyte microinjection and in vitro transcription with DNA molecules methylated in vitro also confirm this conclusion (recently reported at the 1981 Annual Genetics Meeting in Koln, Germany). Agents that can disrupt DNA methylation in vivo can cause diverse effects such as alterations in the pathway of differentiation and reactivation of genes residing in the inactive X chromosome (Taylor and Jones, Cell 7 7, 771-779, 1979; Mohandas et al., Science 27 7, 393-396, 1981). If, in fact, methylation can modulate gene expression, it is important to understand the factors that determine methylation patterns in the cells of vertebrate organisms. It was hypothesized that a methylation pattern, once established in somatic cells, could become inherited in progeny cells (Holiday and Pugh, Science 787, 226-232, 1975; Riggs, Cytogen. Cell Gen. 14, 9-25, 1975). Because the CpG dinucleotide, which bears most of the vertebrate methylation, is a simple palindrome, methylation on one strand could direct the methylation on a newly replicated strand through the action of a “maintenance” methylase that recognizes only hemimethylated sites. In this manner, the organism would have at least one mechanism for the stable somatic inheritance of methylation patterns. Such phenomena as maintenance of X chromosome inactivation and the stability of the differentiated phenotype could be explained in this way. In recent years, evidence has been obtained in favor of this model. Bird (JMB 7 78, 49-60, 1978) studied the distribution of methylation in the ribosomal genes of Xenopus laevis red blood cells. These genes exist in a highly methylated state. They are virtually resistant to digestion with Hpa II, although a few unmethylated Hpa II sites are randomly distributed within these genes. The sensitivity to Hpa II digestion of denatured and reannealed ribosomal genes indicated that virtually all methylated sites were symmetrically methylated, thus suggesting the action of a maintenance methylase of the type postulated above. The amplified ribosomal genes of Xenopus, however, are not methylated (Dawid et al., JMB 57, 341-360, 1970; Bird and Southern, JMB 7 18, 27-47, 1978) which suggests that the specificity for methylation does not reside in the DNA sequence immediately flanking a potential site. Similarly, the endogenous mouse mammary tumor virus sequences are heavily methylated but the integrated sequences arising by horizontal infection are not methylated (Cohen, lot. cit.). Finally, direct evidence for the passive maintenance of methylation patterns comes from two recent studies that have utilized the techniques of DNA-mediated transformation in cultured mouse cells (Pollack et al., lot. cit.; Wigler et al., lot. cit.). DNA molecules were methylated in vitro with the bacterial modification enzyme M-Hpa II, which methylates the internal cyto-

Studies on the miscoding properties of 1,N6-ethenoadenine and 3,N4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis.

1,N6-Ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) are formed when electrophilic vinyl chloride (VC) metabolites, chloroethylene oxide (CEO) or chloroacetaldehyde (CAA) react with adenine and cytosine residues in DNA. They were assayed for their miscoding properties in an in vitro system using Escherichia coli DNA polymerase I and synthetic templates prepared by reaction of poly(dA) and poly(dC) with increasing concentrations of CEO or CAA. Following the introduction of etheno groups, an increasing inhibition of DNA synthesis was observed. dGMP was misincorporated on CAA- or CEO-treated poly(dA) templates and dTMP was misincorporated on CAA- or CEO-treated poly(dC) templates, suggesting that epsilon A and epsilon C may miscode. The error rates augmented with the extent of reaction of CEO or CAA with the templates. Base-pairing models are proposed for the epsilon A.G. and epsilon C.T pairs. The potentially miscoding properties of epsilon A and epsilon C may explain why metabolically-activated VC and its reactive metabolites specifically induce base-pair substitution mutations in Salmonella typhimurium. Promutagenic lesions may represent one of the initial steps in VC- or CEO-induced carcinogenesis.

Hypomethylation of DNA in Raji cells after treatment with N-methyl-N-nitrosourea.

Human Raji lymphoblast-like cells were propagated in the presence of various concentrations of N-methyl-N-nitrosourea (MNU) and the degree of enzymatic methylation of newly synthesized DNA was analysed by two independent methods. The overall extent of enzymatic DNA methylation was measured on the basis of [14C]deoxycytidine derived radioactivity incorporated into DNA 5-methylcytosine and cytosine residues. Enzymatic methylation of internal cytosines at 5'-CCGG-3' sequences of Raji DNA was analysed by use of the bacterial restriction enzyme HpaII and its isoschizomer MspI. The data obtained by both methods indicate that the treatment with MNU causes a lower level of enzymatic methylation of newly synthesized DNA. This lower extent of DNA methylation persists in the absence of the carcinogen in the cell cycles following the treatment.

Comparison of DNAs ofCrypthecodinium cohnii-like Dinoflagellates from Widespread Geographic Locations*

SYNOPSIS. We have examined various properties of DNAs from 7 dinoflagellate isolates of wide geographic distribution; all of the isolates are superficially indistinguishable from a laboratory strain of Crypthecodinium cohnii originally isolated at Woods Hole, Massachusetts (WHd strain). Two isolates, one from Puerto Rico and the other from Honduras, are clearly distinguishable from WHd and the other isolates by their DNA buoyant density values. WHd and the other 5 isolates we have examined are indistinguishable from one another in terms of DNA buoyant densities and melting temperatures. The relationship among the various isolates, including WHd, were evaluated at a finer level through restriction endonuclease cleavage and molecular hybridization to compare ribosomal RNA gene structure in the several DNAs. All the isolates could be further categorized by this method, the patterns of restriction endonuclease cleavage of ribosomal RNA genes in the isolates paralleling exactly their sexual compatibilities established from breeding experiments by Beam & Himes. The DNAs were also treated with a restriction endonuclease sensitive to the presence of the modified base 5‐methylcytosine. In all isolates, cytosine residues in both total DNA and DNA specifically containing the ribosomal RNA genes were found to be extensively methylated, as was previously shown for the WHd strain.

A cautionary note on the use of certain restriction endonucleases with methylated substrates

Methylation of adenine and cytosine residues in DNA isolated from common strains of Escherichia coli K-12 can render that DNA resistant to cleavage by certain restriction endonucleases at those sites at which the recognition sequence for such an endonuclease overlaps (but does not include) a sequence recognized by methylases specified by the dam or dcm gene.

5-methylcytosine formation in wheat embryo DNA

Summary The methylation of cytosine residues in wheat DNA was studied in isolated embryos during the first 30 hrs of germination. L-Methionine, but also L-serine serve as methyl group donors in vivo . DNA methylation reaches high values after the maximum of DNA replication at 18 hrs. Isolated nuclei contain S-adenosyl methionine-dependent methyltransferase activity at all stages of germination, a significant increase is observed after the 22nd hour. The plant enzyme can methylate thymus DNA in vitro , it is inhibited by S-adenosyl methionine analogs.