Digestion of highly modified bacteriophage DNA by restriction endonucleases

Huang, Li Yen Mae; Farnet, Chris M.; Ehrlich, Kenneth C.; Ehrlich, Melanie

doi:10.1093/nar/10.5.1579

Cited by 115 publications

(87 citation statements)

References 44 publications

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“…m) Type III restriction endonuclease (1,26). n) There is a report that Hin C II does not cut GTYRAmC (27). p) There is a report that Xma I does not cut CCmCGGG (8).…”

Section: Introductionmentioning

confidence: 99%

The effect of site specific methylation on restriction endonuclease digestion

McClelland

Nelson

1985

Nucleic Acids Research

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“…m) Type III restriction endonuclease (1,26). n) There is a report that Hin C II does not cut GTYRAmC (27). p) There is a report that Xma I does not cut CCmCGGG (8).…”

Section: Introductionmentioning

confidence: 99%

The effect of site specific methylation on restriction endonuclease digestion

McClelland

Nelson

1985

Nucleic Acids Research

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“…For example, in the cases of Streptomyces achromogenes (Sacd, GAGCTC; SacIl, CCGCGG [30]) and X. campestris pv. oryzae (XorI, (18). Although almost all restriction endonucleases are inhibited by complete substitution of cytosine residues in their DNA by m5C residues (18), that is not considered in this table because the extensive nature of such substitution might have indirect effects on enzyme-DNA interactions.…”

Section: Resultsmentioning

confidence: 99%

N4-methylcytosine as a minor base in bacterial DNA

Ehrlich¹,

Wilson²,

Kuo³

et al. 1987

J Bacteriol

100

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The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.Both N6-methyladenine (m6A) and 5-methylcytosine (m5C) have been long known to be minor bases in bacterial DNA (7,9,16). One or both of these methylated bases are present in most bacterial DNAs examined (4,11,33). Recently, a third minor base, N4-methylcytosine (m4C), has been found in DNA from eight types of thermophilic bacteria (11) and in that from one type of mesophilic bacteria (19,20). Previously, m4C residues had been detected only in the RNA of the small ribosomal subunit of bacteria and of mammalian and insect mitochondria (8,13,40). This modified base is not present in a variety of eucaryotic DNAs (13; Gehrke et al., unpublished results).The minor base composition of bacterial DNA is partially determined by restriction-modification systems (30). The level of minor bases should be consistent with the known specificities of the host restriction and modification enzymes. Also, it can reveal the presence of new DNA methyltransferases. For example, if the known restriction enzymes in a bacterium have recognition sites containing only G. C base pairs and if m6A as well as m5C is found in this cellular DNA, then one or more other unrelated modification pathways must be present. These could be involved in previously unidentified restriction-modification systems in the bacterium or in the control of various DNA functions (3,15,29,35).The present study demonstrates that genomic m4C is not mostly limited to the DNA of thermophilic bacteria. Rather, it is present as a minor base in the DNA of many bacterial mesophiles. Of the 26 bacterial species examined in this study, 9 contained m4C in their genomes. Most of the examined bacteria are mesophiles, and all of them are sources of commercially available restriction endonucleases. The prevalence of m4C as a minor base in these bacterial DNAs indicates that many restriction endonucleases may be inhibited by N4-methylation of cytosine residues in their recognition sites in vivo and that bacterial DNA cytosine methyltransferases must be carefully checked to determine whether they catalyze the formation of m4C. MATERIALS AND METHODSStrains. All bacteria came from the New England BioLabs strain collection except Xanthomonas campestris pv. oryzae, which was from the collection of M. Ehrlich. The * Corresponding author. strains are listed in Table 1 with the sources from which they were originally obtained and the temperature at which the cultures were grown.Purification of bacterial DNA. DNA was prepared from 10 g of wet packed cells suspended in 20 ml of 25% sucrose-50 mM Tris hydrochloride (pH 8.0), and then 10 ml of 0.25 M disodium EDTA (pH 8.0) and 6 ml of 10-mg/ml lysozyme in 0.25 M Tris (pH 8.0) were added. After 2 h at 0°C, 24 ml of 1% Triton X-100 in ...

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“…Methylation of DNA is known to be responsible for poly(dG-m'dC) . poly(dG-m'dC) assuming, under physiological conditions, a left-handed helical conformation known as Z-DNA [42], which unlike B-DNA is highly after prolonged heating of DNA at various ionic strengths (unpublished data), and no evidence for Z-DNA was found in XP12 DNA by X-ray fiber crystallography [54]. Nonetheless, hypermethylated, native XP12 DNA may still possess some different conformation from that of DNAs of normal base The assistance of Mrs C. Perret in the preparation of the manuscript is gratefully acknowledged.…”

Section: Discussionmentioning

confidence: 99%

The accessibility of 5-methylcytosine to specific antibodies in double-stranded DNA of Xanthomonas phage XP12

Adouard¹,

Dante²,

Niveleau³

et al. 1985

Eur J Biochem

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Antibodies specifically directed to 5-methylcytidine were raised in rabbits and purified by affinity chromatography. The accessibility of 5-methyldeoxycytidine (m'dcyd) to such antibodies was studied with DNAs from various origins. The reaction was followed by measuring the retention of radiolabelled DNA by antibodies on nitrocellulose filters, by immunoprecipitation, by gel filtration and was visualized with the electron microscope. Antibodies did not bind to Escherichiu coli B DNA, which is deficient in m'dCyd. Denatured and native DNA from calf thymus, which contains m'dCyd as a minor nucleoside, was weakly retaincd on the filters whereas DNA extracted from Xanthomonus oryzae XP12 bacteriophage, which is rich in m'dCyd, was well recognized even in the native form.Several lines of evidence suggest that methylation of cytosine residues at the 5 position in DNA plays an important role in the mechanisms which control the expression of eukaryotic genes [l -71. The amount of 5-methylcytosine (m'Cyt) in total DNA or DNA fragments can be quantified by thin-layer chromatography or high-performance liquid chromatography [8 -121. These methods allow the detection of small amounts of the modified nucleoside but require chemical or enzymatic hydrolysis of the polynucleotide and do not provide information on the location of m'Cyt on individual molecules. Digestion of DNA with restriction endonucleases sensitive to methylation of CpG sequences, the main site of m'Cyt in vertebrate DNA, and visualization of specific gene regions by blot hybridization with radiolabelled cloned DNA probes [I, 13, have revealed that tissue-specific differences in methylation of cytosine residues are common. However, restriction enzymes sensitive to methylation will only recognize m'Cyt occurring in d(CpG) sequences at certain oligonucleotide sites. Another approach for analysing the distribution of m'Cyt in DNA utilises iinmunochemical methods. Specific antibodies were widely used to demonstrate the presence of methylated bases in RNA, DNA and chromosomes [18]. For DNA, specific antibodies were used mostly to detect m'Cyt either on DNA fragments imniobilised on nitrocellulose paper by Southern transfer [19] or on fixed metaphase chromosomes [20]. Available antisera to 5-methylcytidine (m'Cyd) were shown to react with chromosomes only after ultraviolet irradiation or photooxidation. It had been concluded that the accessibility of m5Cyt residues to antibodies was limited to denatured DNA [21]. Abbreviations. m'Cyt, 5-methylcytosine; m'Cyd, 5-methylcytidine; m'dCyd, 5-methyldcoxycytidine; dCyd, deoxycytidine; HPLC, high-performance liquid chromatography; kb, lo3 base pairs.Enzynfe. Nuclease S1 (EC 3.1.30.1).Here we report results obtained with antibodies directed to m5Cyd and reacted with native or denatured DNAs from various origins. The very sensitive filter-binding method [22] was used to measure the reactivity of these antibodies with bacteriophage XP12 DNA, in which virtually all cytosine residues are methylated [23], with calf thymus DNA in...

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Digestion of highly modified bacteriophage DNA by restriction endonucleases

Cited by 115 publications

References 44 publications

The effect of site specific methylation on restriction endonuclease digestion

The effect of site specific methylation on restriction endonuclease digestion

N4-methylcytosine as a minor base in bacterial DNA

The accessibility of 5-methylcytosine to specific antibodies in double-stranded DNA of Xanthomonas phage XP12

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