Antibodies to Z-DNA interact with form V DNA

Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to Rband-positive heterochromatin, which has a high G+C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R-and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.Repeating sequences of alternating dC and dG deoxynucleotides can form the left-handed duplex DNA conformation, named Z-DNA, as demonstrated by x-ray diffraction studies of crystals of (dC-dG)n (1-3) and fibers of poly (dG-dC)-poly (dG-dC) ( (23, 24), the comparison of the staining patterns produced by antibodies to Z-DNA in euchromatin may be considered a check of the stability of Z-DNA conformation during chromosome evolution.In this work we show (i) that anti-Z-DNA antibodies bind to metaphase chromosomes of man and Cebus in a distinct and specific way and (ii) that an undisputable correspondence exists between the antibody binding and the chromosome banding patterns. In euchromatin, a weak but distinct and consistent staining is observed in the segments corresponding to the Rand T-bands, which are thought to be richer in G-C base pairs both in man and Cebus. Slight differences of intensity are observed from band to band: they are the same for all of the chromosomal segments of man and Cebus, which were found homologous by banding pattern and by gene mapping comparison (23)(24)(25). This fact underlines the stability of Z-DNA conformation during the evolution of mammalian chromosomes. Furthermore, the additional heterochromatic segments existing in Cebus chromosomes exhibit a consistent and very strong binding to anti-Z-DNA antibodies.MATERIAL AND METHODS Obtention of Metaphase Chromosomes. Metaphase cells were obtained from tissue cultures after skin biopsy of a female of C. albifrons held in captivity at the Museum National d'Histoire Naturelle, Paris.Metaphase cells from human lymphocytes were cultured according to usual methods (26). Cell cultures were treated by colchicine for 1 or 2 hr and then for 10 min with a hypotonic solution of diluted human serum (1:6, vol/vol). Cells were fixed in most of the experiments for at least 40 min with two different solutions (solution I, ethanol/chloroform/acetic acid, 6:3:1, vol/ vol; solution II, ethanol/acetic acid, 3:1, vol/vol). A prolonged fixation (45 min to 48 hr), with only the last fixative solution was used for some human cultures.The following banding methods were used to characterize the chromosomes of the specimen of C. albifrons: R-banding 5890 The publication costs of this article were defrayed in part by page charge payment. T...

supporting

confidence: 57%

Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

Viegas‐Péquignot¹,

Derbin²,

Malfoy³

et al. 1983

“…1 Upper gives the titration of the cc DNA of plasmid pLP32 at natural supercoil density with the purified antibody, as revealed by the nitrocellulose binding assay. No interaction of the antibodye.g., with the linear form of this DNA or with pBR322, lacking the (dC-dG)16 sequence-at natural supercoil density is observed (13,14,34). This indicates the importance of the negative superhelical density for sustaining left-handed Z-DNA at low salt concentration.…”

mentioning

confidence: 99%

“…In a semiquantitative assay the mobility change of ccc DNA, due to the binding of the antibody, in 1% agarose gel electrophoresis was employed (14). The gel contained as buffer 36 mM Tris/30 mM NaH2PO4/1 mM EDTA and was run at 5 V/cm, and the DNA was visualized by UV illumination after staining with 0.5 ug of ethidium bromide per ml.…”

mentioning

confidence: 99%

Isolation of Z-DNA-containing plasmids.

Thomae¹,

Beck²,

Pohl³

1983

A purified, monoclonal antibody, specific for the left-handed Z-form of poly(dG-dC), was coupled covalently to Sephacryl S-1000 beads. Such an antibody column provides a convenient method to isolate and purify those plasmid DNAs that contain Z-DNA from a large excess of other DNAs, RNA, etc. From a library of Escherichia coli DNA, cloned into the vector plasmid pUC-8, several recombinant plasmids were isolated, which bind to this antibody. Thus, E. coli contains sequences, which in "natural" negatively supercoiled DNA, adopt a left-handed Z-DNAlike conformation.Physicochemical studies of poly-and oligo (dC-dG (17), were used for cell fusion (22, 23). Cell supernatants were screened for precipitating 32P-labeled poly(dGdC) in 4 M NaCl. Seven monoclonal cell lines producing antibodies to Z-DNA were established (unpublished data). Antibodies (K/y2b) from ascites fluids, by using cell line Z-DI1, were purified by protein A-Sepharose (Pharmacia) chromatography (24, 25). Antibody concentration was determined, by using A280 = 1.4 for a solution at 1 mg/ml. l"I-Labeled antibodies with -10,000 cpm/ng were prepared by the Chloramin-T method (26).Antibody Column. Sephacryl S-1000 (superfine; Pharmacia) was activated with cyanogen bromide in sodium carbonate buffer (27) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…In view ofa possible biological role ofZ-DNA and with respect to the molecular mechanisms involved, the time dependence of the transition in torsionally stressed DNA is of interest. With monoclonal antibodies (mAbs) specific for Z-DNA, a convenient tool is available for measuring the amount of left-handed DNA in the presence of large amounts of B-DNA (12)(13)(14).…”

mentioning

confidence: 99%

Dynamics of the B-to-Z transition in supercoiled DNA.

Pohl¹

1986

The sequence (dC-dG)16, inserted into the polylinker of plasmid pUC8, adopts a lefthanded Z-DNA conformation at "natural" supercoil density. The radioactively labeled monoclonal antibody Z-D11, which has a very high affinity for this DNA conformation, provides a convenient sensitive tool to measure selectively the amount of Z-DNA. Chloroquine reversibly changes the supercoil density of plasmid DNA and thereby the equilibrium between right-and left-handed double-helical DNA. The time-dependent formation or disappearance of Z-DNA was measured by using the antibody either as a fast indicator of Z-DNA or as an additional effector of the B-to-Z equilibrium. In the middle of the transition, a relaxation time of about 1 hr is observed in 0.1 M NaCl at 220C. The kinetic data are compatible with an all-or-none transition between the two conformations. The overall rate constant for Z-DNA formation, kBz, decreases with the square of the chloroqulne concentration, while the reverse one, kzB, increases with about the fourth power.The change between the right-handed B form and the lefthanded Z form of DNA has become one of the best-studied examples for the conformational flexibility of these molecules (for a recent review see ref. 1). Earlier experiments used high sodium chloride concentrations to shift the equilibrium between the two forms (2, 3). The kinetics of the transition in linear double-helical molecules of different length with the sequence (dC-dG). were followed by spectroscopic methods (3). Although such an approach should also be feasible for supercoiled DNA (4,5), the small amount of Z-DNA in plasmids creates considerable experimental problems.Covalently closed circular DNA, as isolated, e.g., from Escherichia coli, has a deficiency of helical turns, which is compensated by a negative supercoiling ofthe molecule (e.g., see ref. 6). Such a DNA is in a state of high energy and a process that relaxes the supercoil density will be favored. One such process is the preferred binding of intercalating molecules, such as ethidium, to covalently closed circular (ccc) DNA, as compared to relaxed or linear DNA (7,8). Another process is a change from a right-to a left-handed double-helical structure (9). The equilibrium of the B-to-Z transition of cloned (dC-dG). in plasmid DNA has been measured mainly by gel electrophoresis (e.g., refs. 10, 11, and 16).The elegant use and analysis by two-dimensional gel electrophoresis has provided a wealth of quantitative information on the energetics of this transition in ccc DNA (11).In contrast, there are practically no quantitative data available on the dynamics of this process in supercoiled DNA. In view ofa possible biological role ofZ-DNA and with respect to the molecular mechanisms involved, the time dependence of the transition in torsionally stressed DNA is of interest. With monoclonal antibodies (mAbs) specific for Z-DNA, a convenient tool is available for measuring the amount of left-handed DNA in the presence of large amounts of .The use of the mAb Z-D11 for isolating ...