Effect of 5-azacytidine on E. coli cells with different DNA-methylases

A correlation was found between the bacteriocide effect of 5-aza-C and the amount of cytosine DNA-methylases in E. coli cells. 5-Aza-C-DNA induced partial or complete inhibition of bacterial DNA-methylases with different site specificity; cytosine DNA-methylases were inhibited by the DNA more effectively than adenine DNA-methylase Eco dam. The inhibitory influence of 5-aza-C-DNA on cytosine DNA-methylases was due to the formation of stable inactive complexes between the enzyme and the non-methylating cytosine analog in the recognition sites. Cytosine DNA-methylase Eco RII formed a relatively firm bond with 5-aza-C-DNA, which could be disrupted by 1 M KCl; this disruption restores the DNA-methylase activity and the inhibiting capacity of 5-aza-C-DNA. Thus, the binding of cytosine DNA-methylase to 5-aza-C in DNA is noncovalent; the inhibition of the enzyme by 5-aza-C-DNA is reversible.     

DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation]

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Tissue specificity of the decrease of cattle lymphocyte DNA methylation during chronic lymphoid leukemia]

It has been found that the content of m5C in the DNA preparations tested have been revealed. The DNAs from normal and leukemic lymphocytes of blood, lymphonodi and spleen differ in ther acceptor ability in the reaction of heterologous methylation in vitro, induced by DNA-methylase from Enterobacter cloacea in the presence of [3H-methyl]S-adenosyl methionine: the ratio of radioactivities in methylated cytosine and adenine residues (m5C/m6A) in leukemic lymphocyte DNA is much lower than in healthy animals' lymphocytes. The decrease in the methylation of DNAs from various lymphoid organs of animals with chronic lymphoid leukemia is well correlated with the impairment. No significant changes of the m5C level and the acceptor ability of the in vitro reaction of heterologous methylation of cow lymph lymphocyte DNA have been observed. The data obtained may be interpreted in terms of tissue (cell) specificity or differences in the degree of DNA methylation under conditions of chronic lymphoid leukemia. It is assumed that the changes in DNA methylation may underlie the disturbances in the regulation of activity of the leukemic cell genetic mechanisms.     

Stimulation of rat kidney, spleen and brain DNA-(cytosine-5)-methyltransferases by divalent cobalt ions

Rat kidney, spleen, brain, and liver DNA-methylases were partially purified by chromatography on DEAE-Trisacryl columns and their catalytic properties were studied. Crude extracts contain one or several inhibitors which are thermostable and resistant to acidic or alkaline treatments and which can be eliminated by dialysis, or by chromatography on DEAE-Trisacryl. These are most probably divalent ions, such as, Pb2+, Zn2+, Cu2+, Fe2+, Mg2+, Mn2+ or Ca2+, which inhibit the DNA-methylase activity. However, Co2+, at concentrations ranging from 0.05 mM to 1 mM, has an efficient stimulatory action on spleen, kidney or brain DNA-methylase activity. The spleen DNA-methylase activity on chicken erythrocyte DNA could be increased 10-fold, by a 0.2 mM concentration of Co2+, but no stimulation was found with liver DNA-methylase. The fact that significant differences exist between the DNA-methylases from the different organs in their behavior towards Co2+ could indicate that these enzymes are different.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.     

Properties of DNA-methylase reaction in isolated nuclei of normal and regenerating rat liver]

Evidence from comparative determination of DNA radioactivity methylation degree of acidic extraction and chlorophormic deproteination of the samples suggest that the former technique is a more efficient one. The properties of the DNA-methylase reaction in isolated rat liver nuclei were studied. The DNA-methylase activity is found to be considerably stable during incubation of the nuclei at 37 degrees C; a broad pH-optimum in the alkaline region is observed (pH 8.6--9.8); this activity is inhibited by Mn2+, nucleotides, actynomycin and S-adenosyl methionine analogs and is activated by Mg2+; the incorporation of methyl groups into DNA is reversible. The data suggest that the DNA-methylase activities of the nuclei isolated at different stages of regeneration do not show substantial variations. No differences in DNA methylation before and after DNA synthesis in the regenerating nuclei were observed. Inhibition of DNA synthesis in the course of regeneration does not decrease the level of DNA methylation. The interrelationship between methylation and replication of DNA is discussed.     

Isolation, purification and properties of restrictase and methylase BstN1 from Bacillus stearothermophilus

Restriction-methylation enzymes BstN1 from Bacillus stearothermophilus were isolated and purified. These enzymes are related to a new class of restriction-methylation enzymes of the second type, whose modifying component is N4-cytosine-DNA-methylase. Both enzymes recognize the DNA sequence CC(A/T)GG. Restrictase BstN1 is a protein made up of one subunit with a molecular mass of 25 kDa. The molecular mass of native DNA-methylase BstN1 is about 55 kDa. The temperature optima for restrictase and methylase BstN1 are around 60 degrees C. Possible uses of BstN1 restriction-methylation enzymes for the analysis of cytosine methylation in bacterial and higher plant DNA are discussed.     

Study of the conditions of activation and stabilization of DNA-methylases from Shigella sonnei 47 during fractionation, purification and storage

A comparative study of activation factors and stabilization conditions of partially purified and individual fractions of DNA-methylases of Shigella sonnei 47 was carried out. The stability of DNA-methylases in the course of storage was examined. The influence of activating factors and stabilization conditions differed significantly depending on the degree of purification and composition of methylase preparations. It was shown that glycerol is ineffective as a stabilizing agent. The activating effect of Ca2+ on Shigella sonnei 47 DNA-methylases was found to be universal, while albumin was shown to exert a more potent stabilizing action. The inactivating effect of proteases on DNA methylation enzymes during storage was demonstrated. A phenomenon of spontaneous fluctuations in the methylating activity of enzymatic preparations of Shigella sonnei 47 upon storage was observed.     

Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.     

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos

Nuclei isolated from the developing sea urchin embryo $\text{Paracentrotus lividus}$ and incubated in the presence of [³H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.     

The sequence specificity of vertebrate DNA methylation.

The relative quantity of 5-methyl cytosine in vertebrate nuclear DNA shows species and tissue variation. To determine whether this is due to the action of species or cell specific DNA methylases the sequence specificity of the 5-methyl cytosine distribution in the DNA of a range of cells has been partially characterised. The pattern of methylation was found to be remarkably constant and indicates stringent evolutionary conservation of the characteristics of vertebrate DNA methylation.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.     

Isolation and various properties of soluble and membrane-bound acid RNAse from rat brain lysosomes

Preparations of soluble (I) and membrane-bound (II) acid RNAse with Mr 68,000 and 72,000 Da, respectively, and purified about 2000-fold were isolated from lysosome-rich fractions of rat brain large hemispheres. RNAase II differed from RNAase I by a lower temperature stability. The pH optimum (pH 5.8-6.1), temperature optimum and substrate specificity of RNAase I and II appeared to be identical. The Km values of RNAases I and II for poly(U) are 166 and 160 micrograms/ml; those for RNA--1200 and 1250 mu k/ml, respectively. RNAases I and II extensively hydrolyze soluble, polymeric RNA, rRNA from brain and yeast and poly(U) but do not influence poly(C), poly(A), poly(G), tRNA and DNA. Monovalent cations (K+, Na+, NH4+) activate both RNAase forms.     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

Factors of activation and stabilization of DNA-methylases from Shigella sonnei 47 and Mycobacterium smegmatis butyricum cells

A comparative study of factors of activation and stabilization of individual DNA-methylases from two bacterial strains--Shigella sonnei 47 and Mycobacterium smegmatis butyricum--isolated by isoelectrofocusing in a pH gradient has been carried out. Storage of enzymes at +4 degrees C (pH 7.5) is accompanied by periodic changes in the methylating activity. No such changes are observed when the enzymes are stored at pI of the protein. In this case the methylases with alkaline or close to neutral values of pI remain stable over a period of at least two weeks, whereas acidic proteins are irreversibly inactivated by the end of a two-week period. A stabilizing effect of BSA on DNA-methylases of Sso 47 and Mbu strains has been demonstrated. A direct correlation between the stabilizing effect of BSA and the degree of enzyme purity has been established. Ca2+ appear to be a universal activator of methylases of the above strains; these cations produce a potent, although a short-term effect and can be used in the production of enzyme preparations with a high specific activity in DNA recombinant technology. Protease inhibitors do not exert any appreciable effect on the methylase activity upon storage. Storage at -20 degrees C and at neutral pH leads to complete inactivation of all DNA-methylases within 24 hours. In this case glycerol is fairly ineffective as a stabilizing agent.     

A new DNA methyltransferase M.BstC8I forms 5'-G(M5C)NNGC-3'. Studying of restriction endonuclease sensitivity to M.BstC8I-methylation

Bacillus stearothermophilus C8 was grown up on the Luria agar at 37 degrees C. A new DNA-methylase was determined in cellular lysate. The methylation of the DNAs of bacteriophages lambda and T7 in the region of 5'-G(m5C)NNGC-3' blocked the activity of BstC8I. Specificity of M.BstC8I was analyzed on methylated lambda DNA. For this purpose, we used computer modeling and the data on the sensitivity of restrictases BstC8I, BsuRI, AjnI, and PvuII to methylation. The sensitivity of some restrictases to new methylation was studied. The results may be used for DNA methylation studying.     

Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.