Calf Thymus dsDNA Research Articles

Detection of Chemically Induced DNA Damage by Derivative Square Wave Voltammetry

Damage of DNA films after reaction with styrene oxide was detected using derivative square wave voltammetry. Double-stranded (ds) DNA films with initially low backgrounds developed oxidation peaks for DNA bases during incubation with styrene oxide. Films were prepared on pyrolytic graphite (PG) electrodes by casting mixtures of DNA with the poly(ester sulfonic acid) ionomer Eastman AQ38S or by covalent binding of DNA onto oxidized PG. While both types of films gave oxidation peaks in the region 0.6-1.1 V vs SCE after incubations with styrene oxide, DNA/AQ films gave the best signal-to-background ratios. Damage of DNA by reaction with styrene oxide under the electrode incubation conditions was confirmed by capillary electrophoresis. Total integrals of oxidation peaks increased with time of incubation with styrene oxide. Relative peak heights depended on the type of DNA in the order calf thymus ds DNA > salmon sperm ds DNA > supercoiled ds DNA > highly polymerized calf thymus ds DNA.

DNA quadruplexes assembled by simple peptide: effects of DNA homology and peptide removal.

Formation of heterologous (calf thymus dsDNA) and homologous (linearized pBR322 plasmid dsDNA) quadruplexes upon binding with the simple aliphatic tripeptide derivative (L-Val)3-N2H2-DNS.CF3COOH-DHTV) was examined by fluorimetry, flow linear (LD), circular dichroism (CD), and electron microscopy (EM). The morphology of the rod-like compact particles formed due to the association of dsDNA segments proved to be the same for both DNAs, whereas the stability of the compact DNA structure upon tripeptide removal from the complex with DNA differed substantially for homologous versus non-homologous dsDNA used. The increase in NaCl concentration in the solution up to 30 mM removes the peptide from both types of the complexes completely. At the same time at 20 mM NaCl calf thymus DNA quadruplexes readily dissociate, whereas the structures formed by plasmid DNA retain their morphology in the solution containing NaCl with concentrations up to 40 mM and are only partially disrupted at even higher NaCl concentration. These results provide an analogy between trivaline-DNA model complexes and RecA-DNA binding.

Modulation of renal disease in autoimmune NZB/NZW mice by immunization with bacterial DNA.

Preautoimmune New Zealand Black/White (NZB/NZW) mice immunized with Escherichia coli (EC) double standard (ds) DNA produce antibodies that bind mammalian dsDNA and display specificities similar to spontaneous lupus anti-DNA. Since calf thymus (CT) dsDNA fails to induce these antibodies, these results suggest a special potency of foreign DNA in inducing serological manifestations of lupus in a susceptible host. To assess the effects of DNA immunization on clinical manifestations in NZB/NZW mice, we measured renal disease and survival of mice immunized with either (a) EC dsDNA as complexes with methylated bovine serum albumin (mBSA) in adjuvant; (b) CT dsDNA with mBSA in adjuvant; (c)mBSA alone in adjuvant; or (d) unimmunized. After immunization with EC dsDNA, NZB/NZW mice developed significant levels of anti-dsDNA antibodies. Nevertheless, these mice had less proteinuria, nitrate/nitrite excretion, and glomerular pathology than mice immunized with either mBSA alone, CT dsDNA/mBSA complexes, or unimmunized mice. Survival of the EC dsDNA immunized mice was significantly increased compared with the other mice. Furthermore, immunization of mice after the onset of anti-DNA production and proteinuria stabilized nephritis and prolonged survival. The improvement in renal disease occurred despite the expression of autoantibodies that bound mammalian dsDNA as well as glomerular antigens. These results suggest that bacterial DNA has immunological properties that attenuate murine lupus despite the induction of pathogenic antibodies.

Lupus autoantibodies to double-stranded DNA cross-react with ribosomal protein S1.

One cDNA clone (G7) was isolated from a lambda gt11 human liver cDNA library by the reaction with a serum containing anti-dsDNA Abs and was ligated into pGEX-1 lambda T vector. All the 10 SLE sera with anti-dsDNA, 2 samples of human monoclonal anti-dsDNA (33.C9 and 33.H11), and 2 affinity-purified anti-dsDNA Abs recognized the glutathione S-transferase fusion protein expressed by G7 (G7-FP). Ab binding to the recombinant protein expressed by G7 (G7-RP) and to G7-FP was inhibited completely by calf thymus dsDNA. The cDNA was 1314 nucleotides in length and contained an open reading frame encoding 352 amino acids. However, it seems to be a partial length cDNA because the affinity-purified Ab from G7-FP recognized only a 104-kDa protein on Western blot using MOLT4 cell extract. The nucleotide sequence of G7 was homologous (99% identity) to a cDNA encoding human ribosomal protein (r-protein) S1 homologue mRNA. The encoded protein contains repeating residues as a feature of r-proteins S1. Cytoplasmic and nucleolar staining of 33.H11 on indirect immunofluorescence (IF) using HEP 2 cells was inhibited by both G7-RP and calf thymus dsDNA. On ELISA, 33.H11 had a higher affinity for G7-RP than for DNA while 33.C9 had a higher affinity for DNA than for G7-RP and binds nuclei on IF. We conclude that G7 encodes a portion of human r-protein S1 and anti-dsDNA Abs cross-react with this protein.

Molecular analyses of anti-DNA antibodies induced by polyomavirus BK in BALB/c mice.

In the present experiments, two groups of BALB/c mice (five individuals in each group) were hyperimmunized through four consecutive immunizations with either BK virus (Group 1) or BK dsDNA complexed with methylated BSA (Group 2). All immune sera taken after the fourth immunization from both groups reacted strongly with polyomavirus BK dsDNA as well as with calf thymus dsDNA, and all sera contained antibodies that bound in the Crithidia luciliae assay. This indicates that polyomavirus BK was able to induce antibodies with binding characteristics similar to SLE anti-DNA antibodies. To further characterize these induced anti-DNA responses, 10 monoclonal anti-DNA antibodies (four from Group 1, and six from Group 2) were generated and selected for reactivity with S1-nuclease digested CT dsDNA. Their specificity for BK and CT dsDNA molecules, as well as their light and heavy chain variable region cDNA nucleotide sequences were analysed to compare them with known SLE derived anti-DNA antibodies. All of the 10 antibodies bound strongly to BK dsDNA, while seven also bound to CT dsDNA in competitive ELISA experiments. V-region analysis revealed that the induced antibodies resembled anti-DNA antibodies characteristic for murine SLE, and all but one contained arginine in the VH CDR3 region. The arginines present in the monoclonal antibodies originated either from an RF shift from RF1-->RF3 of the D-genes or from N-sequence additions. Taken together, the data demonstrate that anti-DNA antibodies in response to hyperimmunization with polyomavirus BK have the same characteristics as of those occurring spontaneously in SLE. As virus infection/replication in vivo implies expression of immunogenic (non-self) DNA-binding proteins that may render DNA immunogenic, the present results may therefore suggest one physiological mechanism for production of SLE-related anti-DNA antibodies.

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.

Antibodies to Viral and Mammalian Native DNA in Response to BK Virus Inoculation and Subsequent Immunization with Calf Thymus DNA

Recent studies have demonstrated that anti-DNA antibodies share important genetical features with antibodies to exogenous antigens, suggesting that anti-DNA antibody responses may be (auto-) antigen driven. We have earlier defined three out of five rabbits as anti-dsDNA antibody responders based on reactivity with calf thymus (CT) dsDNA after inoculation with the human dsDNA virus BK. In the present study we demonstrate that all five animals that received BK virus inoculations produced antibodies to BK virus dsDNA. These antibodies did not cross-react with CT dsDNA, as shown by inhibition experiments. The anti-BK dsDNA antibodies persisted over time, in contrast to the anti-CT dsDNA antibodies that decreased shortly after a peak following the first boost of BK virus. While the anti-CT dsDNA antibodies decreased, the anti-BK dsDNA antibodies remained elevated, thus supporting the results of the inhibition experiments which showed that two independent antibody populations are produced after BK virus inoculations. In the three animals producing anti-mammalian dsDNA antibodies, antibodies recognizing CT dsDNA reappeared after intravenous administration of a complex of CT dsDNA and methylated bovine serum albumin (MBSA) without adjuvant. The latter anti-CT dsDNA antibodies did not cross-react with BK dsDNA. In contrast to earlier studies we conclude that mammalian dsDNA may be immunogenic, and that discrete molecular differences in DNA antigens from different sources may induce anti-dsDNA antibodies specific for dsDNA molecules of different origin.

Effect of DNA conformation on the hydroxyl radical-induced formation of 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA.

Reactions of hydroxyl radicals with DNA form a variety of base and sugar products and 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA. Here we report the effect of DNA conformation on the yields of 8,5'-cyclopurine 2'-deoxynucleosides and the ratios of their (5'R)- and (5'S)-diastereomers. Calf thymus DNA in native (double-stranded DNA) or heat-denatured form (single-stranded DNA) was exposed to hydroxyl radicals generated by ionizing radiation in nitrous oxide-saturated phosphate buffer. Doses ranging from 10 to 40 Gy were used to ensure low levels of damage to DNA and thus to preserve its secondary structure in experiments with double-stranded DNA (ds-DNA). After irradiation, DNA was hydrolysed enzymatically to 2'-deoxyribonucleosides. The hydrolysates were dried, trimethylsilylated, and analyzed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. An internal standard was used for quantitative measurements and added to DNA samples prior to enzymatic hydrolysis. The yields of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in single-stranded DNA (ss-DNA) were higher than those in ds-DNA. The (5'R)-diastereomers of both compounds were found to predominate over their (5'S)-diastereomers in ss-DNA. In contrast, the yields of the (5'S)-diastereomers in ds-DNA were slightly higher than those of the (5'R)-diastereomers. The G values of 8,5'-cyclo-2'-deoxyadenosine in ss-DNA and ds-DNA were 0.042 and 0.025, respectively. Those of 8,5'-cyclo-2'-deoxyguanosine in ss-DNA and ds-DNA were 0.038 and 0.017, respectively.

A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-lpr/lpr mouse: specificity and idiotype in serum immunoglobulins

Anti-double stranded (ds) DNA antibodies in the sera of lupus-prone MRL/Mp-lpr/lpr (MRL/1) mice were determined by an enzyme-linked immunosorbent assay in parallel with anti-single stranded (ss) DNA, anti-left-handed Z-DNA and anti-poly(ADP-ribose) antibodies. The serum levels of these antibodies in these mice increased with age, and in particular anti-dsDNA antibodies appeared in mice more than 14 weeks old, along with progressive lymphadenopathy. We therefore established a hybridoma producing monoclonal antidsDNA antibody (2C10) from an 8-month-old MRL/1 mouse. Monoclonal antibody 2C10 did not react with either poly(dT) or poly(I), which are major cross-reactants with previously reported monoclonal MRL mouse autoantibodies. Antibody 2C10 showed preference for φX-174 replicative form DNA and calf thymus dsDNA over ssDNA. 2C10 idiotype (Id) was present in the sera of MRL/1 mice, but only occasionally at high levels even in the aged mice tested. This result suggested that many Ids with anti-dsDNA antibody activity may contribute to lupus pathogenesis in this strain of mouse.

Specificity of anti-DNA antibodies in SLE—I. Definition and gross specificity of antibody populations in human SLE plasma

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations ofanti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonuclcotides (average length tetranucleotides). For one of the plasma tested (PS). pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.