Immunological detection of left-handed Z DNA in isolated polytene chromosomes. Effects of ionic strength, pH, temperature and topological stress.

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

doi:10.1002/j.1460-2075.1984.tb01875.x

Cited by 78 publications

(39 citation statements)

References 71 publications

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“…It has been reported that brief preincubation of cells at low pH enhances the binding of anti-Z-DNA antibodies (17). We performed a series of experiments in which lymphocytes were pretreated with low-pH buffers, neutralized, and then exposed to the primary anti-Z-DNA antibodies (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Staiano‐Coico¹,

Stollar²,

Darzynkiewicz³

et al. 1985

Mol. Cell. Biol.

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Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosphila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (GO or GIQ) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (GI), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from GI through S and G2 is correlated with a '27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).Left-handed Z-DNA has been shown to exist in crystals and can be induced in polynucleotide solutions in certain ionic environments and in supercoiled DNA (9,15,16,20). This and other stabilized forms of Z-DNA are immunogenic, and antibodies specific for Z-DNA have been raised by a number of investigators (10, 11, 13). The anti-Z-DNA antibodies have then been used to identify areas within the DNA molecule that can assume the left-handed form in polytene chromosomes of insects (2, 14) and metaphase chromosomes (19) of mammalian cells by in situ immunofluorescence.In the present study we have used multiparameter flow cytometry to measure the binding of anti-Z-DNA antibodies within the nuclei of quiescent and mitogen-stimulated human peripheral blood lymphocytes in relation to total DNA content. Thus, anti-Z-DNA antibody binding could be mea-

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Section: Resultsmentioning

confidence: 99%

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Staiano‐Coico¹,

Stollar²,

Darzynkiewicz³

et al. 1985

Mol. Cell. Biol.

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“…In the case of nuclear Z-DNA, histone removal apparently induces negative supercoiling and stabilizes alternating purine-pyrimidine sequences in Z-conformations such that they are specifically recognized by anti-Z-DNA immunoglobulins in deproteinized cells (16). The existence of Z-DNA or Z-RNA in vivo has not been shown.…”

Section: Methodsmentioning

confidence: 99%

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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“…DNA sequences that are in the Z conformation (or that may be induced to adopt the Z conformation) have been identified in vivo (3)(4)(5)(6), and a role for Z DNA in the regulation of gene expression has been proposed (1).…”

mentioning

confidence: 99%

Thermodynamics of the B to Z transition in poly(m5dG-dC).

Chaires¹,

Sturtevant²

1986

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

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Differential scanning calorimetry, temperature-dependent absorbance, and circular dichroic spectroscopy were used to study the thermodynamics of the B-Z transition in poly(m5dG-dC). In sodium phosphate buffer (pH 7.0) containing 50 mM NaCl and 1.0 mM MgCl2, the reversible B-Z transition of the polymer is centered at 38.20C and is characterized by AHw = 0.61 -0.07 kcal/(mol-base pair) (1 cal = 4.184 J), AIVH = 68 ± 7 kcal/mol, and a cooperative unit of length 110 ± 20 base pairs. A second transition centered at 53.60C is observed and represents a conformational change of poly(m5dG-dC) from the Z to an alternate left-handed form of unknown structure. This transition does not arise from aggregation to the Ze form, or from the helix-coil transition. The left-handed helix-coil transition is observed at 120.9 ± 0.3OC and is characterized by AHcw = 9.1 -1.0 kcal/(mol-base pair).These results indicate a substantial enthalpic contribution to the B-Z transition, in contrast to previous assertions that the process is entropically driven.The chemistry and biology of left-handed Z DNA are of intense current interest (1, 2). DNA sequences that are in the Z conformation (or that may be induced to adopt the Z conformation) have been identified in vivo (3-6), and a role for Z DNA in the regulation of gene expression has been proposed (1).In spite of a considerable number of fundamental studies on the B-Z transition, the thermodynamics of the process remains incompletely described. In their seminal work on the transition of poly (dG-dC) to what has proven to be the left-handed Z form, Pohl and Jovin (7) reported that the NaCl-induced transition of the polymer was independent of temperature over the range 20-50'C and inferred from their data that the van't Hoff enthalpy of the transition was near zero under their conditions. Roy and Miles (8), however, reported a thermally driven interconversion of poly(m5dG-dC) from the B to the Z form in dilute Na+/Mg2+ solutions. Temperature-dependent conformational transitions to the Z form in poly(dG-dC) and poly(m5dG-dC) under a variety of solution conditions were subsequently reported (9-14).We report here results obtained from differential scanning calorimetry (DSC) that provide a reliable estimate for the enthalpy of the B-Z transition of poly(m5dG-dC). DSC measurements provide a direct and sensitive measure of the transition enthalpy, and thus offer a significant advantage over methods based on the indirect van't Hoff analysis. Complementary absorbance and circular dichroic measurements allow us to identify the conformation of the polymer as a function of temperature. Our results provide evidence for a previously unobserved conformational transition within the left-handed DNA family. MATERIALS AND METHODSPolynucleotides. Poly(m5dG-dC) (lot 782-26 and lot NG517938) was purchased from Pharmacia P-L Biochemicals. Samples were used without further purification and were prepared by dissolving the polymer in 6 mM Na2HPO4/ 2 mM NaH2PO4/50 mM NaCl/1 mM MgCl2, pH 7.0 (henceforth referred...

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Immunological detection of left-handed Z DNA in isolated polytene chromosomes. Effects of ionic strength, pH, temperature and topological stress.

Cited by 78 publications

References 71 publications

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Cytoplasmic Z-RNA.

Thermodynamics of the B to Z transition in poly(m5dG-dC).

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