Binding of deoxyadenylate and deoxycytidylate antibodies to double-stranded DNA

Antibodies raised against deoxyadenylate and deoxycytidylate were found to react with double stranded DNA as assessed by highly sensitive avidin-biotin microELISA. The binding was specific as it was completely inhibited by the homologous hapten. The antibodies did not react with tRNA and rRNA. These antibodies were also shown to react with supercoiled and relaxed forms of pBR322 DNA as demonstrated by gel retardation assay.     

Base specific fractionation of double stranded DNA: affinity chromatography on a novel type of adsorbant.

A material suitable for base-pair-specific "affinity chromatography" of double stranded DNA is described. The synthesis of this material involves two successive polymerization reactions yielding solid particles of cross-linked bisacrylamide to which base-pair-specific dyes are covalently attached by spacers of polyacrylamide chains of different length. Materials with immobilized A.T-specific malachite green or G.C-specific phenyl neutral red were used for base-pair-specific fractionation of sheared DNA from bacterial sources, as well as of higher molecular weight Calf thymus DNA or defined fragments of coliphage lambda DNA. The excellent resolving power of this method of "affinity chromatography" of nucleic acids is comparable only to DNA fractionation in buoyant density gradients in the presence of base-pair-specific ligands. A great advantage of this material is its simple handling and its chemical stability, which allows repeated re-use of the same column.     

Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient.

The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined. DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay. Antibodies specific to double stranded DNA were elicited. Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli. These results suggest that KS8 DNA has a significant antigenicity in mice.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

Co-localisation of autoimmune antibodies specific for double stranded DNA with procorticotrophin-releasing hormone within the nucleus of stably transfected CHO-K1 cells

Human autoantibodies and corticotrophin-releasing hormone (CRH)-specific antibodies have been used in a double-labelling immunofluorescence technique to demonstrate that immunoreactive CRH structures are co-localised with immunostaining produced by double stranded DNA-specific human autoantibodies within the nucleus of cultured ovarian cells of Chinese hamsters (CHO-K1). This co-localisation was confirmed using confocal microscopy. A metabolic labelling technique was used to investigate the role of the cytoskeleton in mediating nuclear translocation of proCRH within stably transfected CHO-K1 cells and showed that microtubule and actin disrupting agents had no effect upon the nuclear translocation of proCRH. These results, therefore, suggest that nuclear translocation of proCRH is not affected by drugs which disrupt the cytoskeleton and, consequently, modify the diameter of the nuclear pores.This work was supported by proproject grants from the BBSRC to M.G.C. and P.R.L., and an MRC (UK) grant to M.G.C. M.G.C. and P.R.L. would also like to acknowledge the support received from the, The Wellcome Trust, Welsh Scheme for the Development of Health and Social Research, Sir Halley Stewart Trust, The Royal Society, and the Department of Physiology, UWCC. P.R.L. is a Research Fellow from the Lister Institute of Preventive Medicine.     

Biotinylation of double stranded DNA after transamination

The bisulfite catalyzed transamination of cytidine and cytosine has been reported to be single strand specific, but local thermal instabilities of the DNA double helix, coupled with the extreme sensitivity of the Biotin-Avidin revelation methods, allows the random labelling of cytosines in d.s. DNA to detectable levels for those purposes where the overall label can be very low. We have evaluated the use of this reaction to prepare double stranded DNA molecules containing N4-aminoethyl-cytosine (4-aeC). After this step 4-aeC residues can be conjugated to biotinyl-n-hydroxysuccinimide ester yielding biotinylated DNA. This reaction allows the massive production of biotinylated probes. Labelled DNA can serve as molecular weight marker and positive control in Southern-blots. Moreover it can be useful in the study of DNA-protein interaction and in the isolation of d.s. DNA-binding proteins through chromatographic procedures.     

Amphiphilic beta-sheet peptides can bind to double and triple stranded DNA

It was shown that synthetic peptides with amphiphilic beta-sheet structure can bind to and stabilize double and triple stranded DNA. CD spectra indicated that beta-sheet conformation of peptides were emphasized in the presence or absence of DNA and that no significant change of DNA conformation occurred. UV melting study at pH 7.0 revealed that interaction of peptides with DNA and its hybrids are sensitive and specific depending the host structure.     

The preprotachykinin A promoter interacts with a sequence specific single stranded DNA binding protein.

An element within the Preprotachykinin A (PPT) promoter is highly homologous to an element from the rat type II Na channel promoter. This Na Channel element has been previously proposed to be common to a number of neuronal genes. We demonstrate that the PPT element binds a sequence specific DNA binding protein. The protein binds to only one strand of the PPT element and has little or no specificity for the double stranded DNA species. Gel retardation analysis indicates that the protein is found in both rat neuronal tissue and adult dorsal root ganglia neurons in culture but not in established tissue culture cell lines. Using the PPT element linked to magnetic beads we have been able to demonstrate the enrichment of a protein with a molecular weight of 40k with that of the binding activity. A mechanism for protein binding to the DNA is proposed based on the fact that the region binding the protein is the loop of a larger stem-loop structure in the DNA.     

Dynamics of double stranded DNA reptation from bacteriophage.

The dynamics of dsDNA release process from a phage head has been analyzed theoretically. The process was considered as dsDNA reptation through the phage tail. The driving force is assumed to be the decrease of the DNA globule free energy on its releasing from the head in the surrounding medium. The results of the equilibrium theory on an intraphage DNA globule were applied. Three possible sources of friction were examined. The first one is in the inner channel of the tail. The second is the friction of DNA segments in the whole globule volume, which is essential when the globule decondensation is a collective process, at simultaneous moving of all the turns (mechanism 1). The third is the globule friction with the capsid inner surface, that is most important when decondensation proceeds via the globule rotation as a whole spool (mechanism 2). Mechanism 1 would require a lot of time for ejection. Mechanism 2 would lead to different ejection dynamics of short- and long-tailed phages. Comparison of the theoretical results with the published experimental data argues in favor of mechanism 2.     

Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

Cleavage of double stranded plasmid DNA by lanthanide complexes

The use of free lanthanide ions and their complexes for plasmid DNA pBR322 and chromosomal DNA cleavage was studied. Plasmid pBR322 DNA was treated by lanthanide chlorides (Eu(3+), La(3+), Nd(3+), Pr(3+), Gd(3+)) in HEPES buffer (pH 7.0, 7.5 and 8.0) at 24, 37, 50, 63, and 76 degrees C. The formation of linear and nicked plasmid forms was investigated depending on the reaction conditions. Heterogeneous lanthanide complexes of ethylenediamine tetraacetic acid (EDTA) immobilized on insoluble methacrylate support and iminodiacetic acid (IDA) immobilized on styrene support were used as catalysts plasmid for DNA pBR322 cleavage, too. The temperature of reaction mixture had substantial influence on cleavage rate. The precipitation of DNA occurred during the measurement of interactions between chromosomal DNA and La(3+) ions.     

Design and applications of single and double stranded DNA analogs.

Chemical ligation method for assembling modified DNA-duplexes as well as solid support method and postsynthetic modification in aqueous media was used to synthesize a variety of single and double stranded oligonucleotide derivatives. Novel oligodeoxynucleotides with cluster or alternating sugar modifications and probes resistant to cell nuclease degradation were obtained. The use of such probes for effective and regiospecific hybridase RNA-hydrolysis is provided. Effective synthesis of new DNA duplexes containing modified and chemical cleavable internucleotide bonds was developed. A novel type of affinity reagent was suggested for covalent attachment of substrate to active site nucleophiles in the restriction/modification systems.     

Sequence-dependence and prediction of nucleotide solvent accessibility in double stranded DNA

Solvent accessibility of amino acid residues in proteins has been widely studied and many methods for its prediction from sequence and evolutionary information are available. Some of the advantages of studying amino acid solvent accessibility also apply to DNA. However, currently there are no methods to estimate the solvent accessibility of nucleotides, as most works on DNA structures have focused on elastic deformations and other structural attributes. In this work, an attempt has been made to analyze the distribution of different nucleotides in various accessibility ranges. Effect of neighboring nucleotides on the predictability of exposure has been evaluated by developing a linear perceptron model that takes sequence information as the input. Five different types of solvent accessibility (overall nucleotide, side chain, main chain, polar and non-polar) have been predicted. From the analysis, it is observed that Thymine stands out in terms of its higher exposed surface area, particularly its side chain and non-polar atoms. It is also concluded that the solvent accessibility of a nucleotide strongly depends on its sequence neighbors and can be predicted with fair success using this information.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

A nuclease from Neurospora crassa specific for d(A-T) rich regions in double stranded DNA

A nuclease from N. crassa has been prepared to the hydroxylapatite stage of purification described by Rabin and Fraser (1). It degrades single stranded DNA in an essentially exonucleolytic process. It does not give any appreciable acid soluble material with double stranded DNA as substrate. This shows its high degree of specificity towards single stranded DNA.