Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids

Nahon-Merlin, E.; Delain, Etienne; Coulaud, Dominique; Lacour, F

doi:10.1093/nar/8.8.1805

Cited by 15 publications

(9 citation statements)

References 34 publications

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“…In these studies we have used darkfield electron microscopy of stained and unshadowed molecules which have not been embedded in a basic protein film (Nahon-Merlin et al, 1980;Revet and Delain, 1982). These EM techniques offer many advantages over other mapping techniques for localizing DNA-binding proteins on particular nucleotide sequences, because individual members of the native population can be clearly resolved.…”

Section: Advantages Of High Resolution Darkfield Em Of Native Dna-iggmentioning

confidence: 99%

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

Révet¹,

Zarling²,

Jovin

et al. 1984

The EMBO Journal

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The specific interaction between left‐handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti‐Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno‐electron microscopy. DNA‐antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti‐Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti‐Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti‐Z IgG binding sites which were expressed with varying frequencies (53‐3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non‐cooperative due to relaxation of torsional stress by the B–‐Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.

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Section: Advantages Of High Resolution Darkfield Em Of Native Dna-iggmentioning

confidence: 99%

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

Révet¹,

Zarling²,

Jovin

et al. 1984

The EMBO Journal

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“…Using the molecular weight of 80,000-100,000 provided by the manufacturer, and assuming a purely double-helical structure and a base pair molecular weight average of 301, one calculates that the polymer should contain between 265 and 332 base pairs. Conversely, assuming a purely A-form double helix with an axial rise per base pair of 2.82Å (Nahon- Merlin et al 1980) and a poly r(A-U) length ranging from 87-110 nm, it is calculated that the poly r(A-U) should contain 309-390 observed diameters of the poly r(A-U) range from 8.8 -21 nm, which is larger than the expected value of 23 Å (2.3 nm) and is indicative of geometric tip effect. However, these values are consistent with those observed by Zenhausern et al (1992c) with plasmid DNA.…”

Section: Poly R(a-u) Alonementioning

confidence: 99%

Transmission electron microscopy and scanning force microscopy of poly r(A‐U) and poly r(A‐U)‐ethidium bromide

et al. 1997

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Summary:Transmission electron microscopy and scanning force microscopy of negative-stained, carbon-coated replica and mica-adsorbed preparations of 200 µM poly r(A-U) and 50 µM ethidium bromide/200 µM poly r(A-U) have been employed to evaluate ethidium-induced changes in poly r(A-U) topology. Poly r(A-U) alone exhibits elongated conformations 85-115 nm in length that possess a number of hairpin loops as well as single-stranded domains. While the doublestranded domains are found predominately at the base of the hairpin loops (diameter = 5-30 nm), other rod-like (presumably double-stranded) regions ranging from 25-80 nm in length are present in other portions of the poly r(A-U). In contrast with the poly r(A-U) alone, the EB/poly r(A-U) combination appears as a heterogeneous population of condensed structures whose lengths and widths vary from 12-88 nm and 15-45 nm, respectively. These conformational changes are due to a number of factors, including the displacement of ordered water surrounding the poly r(A-U) and charge shielding of the phosphate groups of the poly r(A-U) upon the binding of the ethidium.

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“…After 2 min, grids were stained with 2% aqueous uranyl acetate with previous rinsing with 0.1 M sodium cacodylate buffer, pH 7.4, to minimize precipitation of uranyl acetate with phosphate ions. Grids were either observed directly or after shadow casting with an ultra-thin film of tungsten [24] using the tilted dark field illumination mode with a Philips EM-300 electron microscope.…”

Section: Electron Microscopymentioning

confidence: 99%

“…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 which m'Cyt comprises only 1.4% of the total bases [I21 and with Escherichia coli B DNA which contains no detectable m'Cyt [12]. The binding of antibodies to native DNA from XP12 phage was further demonstrated by immunoprecipitation, gel filtration and electron microscopy using methods previously employed for the study of the binding of antibodies to synthetic copolymers [24]. The results obtained indicate that methylated residues in both double-stranded and singlestranded hypermethylated XP12 DNA were accessible to these antibodies whereas calf thymus DNA was poorly recognized.…”

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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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Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids

Abstract: The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM)

Cited by 15 publications

References 34 publications

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

Transmission electron microscopy and scanning force microscopy of poly r(A‐U) and poly r(A‐U)‐ethidium bromide

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

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