Interactions of Anti-poly[d(G-br<sup>5</sup>C)] IgG with Synthetic, Viral and Cellular Z DNAs

Zarling, David A.; Arndt‐Jovin, Donna J.; McIntosh, Lawrence P.; Robert‐Nicoud, Michel; Jovin, Thomas M.

doi:10.1080/07391102.1984.10507506

Cited by 17 publications

(21 citation statements)

References 63 publications

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“…Furthermore, dimers, trimers, and other multimers were observed. Previously, we have shown that these multimers are formed by the binding of bivalent anti-Z-DNA IgGs to Z-DNA tracts on individual DNA molecules (3,4,7,8). In contrast, there was no reactivity of form I M13 or pBR322 at their extracted negative superhelical densities with C-6, D-4, or normal rabbit IgGs (Fig.…”

Section: Methodsmentioning

confidence: 84%

“…Direct RIA or competition assays with unlabeled polymers or drugs were as described (3)(4)(5) Cytological Preparations, Immunofluorescent Staining, and Photomicroscopy. Crithidia luciliae and Crithidia fasciculata were propagated in culture medium containing 37 mg of brain-heart infusion per ml (Difco) and 25 ,ug of hemin per ml.…”

Section: Methodsmentioning

confidence: 99%

“…The phosphodiester backbone and 2' hydroxyl groups in Z-RNA may be in close enough juxtaposition to specify a 15-to 20-A antibody-binding site (for further discussion, see refs. [3][4][5]. Furthermore, these groups may also be in the vicinity of the exposed portions of the bases on the major convex surface, possibly including the atoms at the 8 position on guanosine and the 5 position on cytosine.…”

Section: Methodsmentioning

confidence: 99%

“…Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-depleted) cellular DNA (3,4). Nuclease cutting, topisomerase relaxation, or treatment with certain intercalating drugs can induce a Z --B transition in plasmid or cellular DNA and eliminate the binding of specific anti-Z-DNA IgG probes.…”

mentioning

confidence: 99%

“…Nuclease cutting, topisomerase relaxation, or treatment with certain intercalating drugs can induce a Z --B transition in plasmid or cellular DNA and eliminate the binding of specific anti-Z-DNA IgG probes. Small amounts of Z-DNA or potential Z-sequences can be detected by anti-Z-DNA IgG immunofluorescence in nuclear DNA from a variety of fixed cells (1)(2)(3)(4). Though it appears that some alternating purine-pyrimidine sequences can be transcribed in nuclear DNA, it is not known whether transcribed RNA can adopt the left-handed Z-RNA conformation.…”

mentioning

confidence: 99%

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Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...

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Section: Methodsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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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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Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Hagen¹,

Zarling²,

Jovin

1985

The EMBO Journal

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Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 itM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Nonimmune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density). Key words: left-handed Z DNA/enhancer/alternating purine-pyrimidine Introduction We have previously developed an electrophoretic assay which detected left-handed Z DNA sites in SV40 DNA (and other circular genomes) using anti-Z DNA IgG binding to negatively supercoiled Form I molecules (Zarling et al., 1984a(Zarling et al., , 1984b. The formation of DNA-IgG complexes depended on external conditions (ionic strength and temperature), nucleotide sequence, the physical state of the DNA (torsional stress) and the anti-DNA immunoglobulin (specificity, valency and concentration). Changes in electrophoretic migration were attributed to the formation of various oligomeric species; the existence of trimers suggested the expression of at least two Z DNA sites per SV40 DNA.In the present study, SV40 Form I DNA-IgG complexes-were preparatively purified by velocity sedimentation using conditions which optimally stabilize natural Z DNA sequences in protein-

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Interactions of Anti-poly[d(G-br⁵C)] IgG with Synthetic, Viral and Cellular Z DNAs

Cited by 17 publications

References 63 publications

Cytoplasmic Z-RNA.

Cytoplasmic Z-RNA.

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

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

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Interactions of Anti-poly[d(G-br5C)] IgG with Synthetic, Viral and Cellular Z DNAs

Cited by 17 publications

References 63 publications

Cytoplasmic Z-RNA.

Cytoplasmic Z-RNA.

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

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Contact Info

Product

Resources

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Interactions of Anti-poly[d(G-br⁵C)] IgG with Synthetic, Viral and Cellular Z DNAs