The reactivity of lupus erythematosus serum with native and denatured DNA of various origins]

The serum of a female suffering from Lupus erythematosus visceralis was investigated by complement fixation for the reaction with native and denatured DNA's of various base composition. The reaction with native DNA is independent on the (G+C)-content of the DNA. It is apparent that the responsible antibodies react with determinants of the helical conformation, which are identical in the various DNA-molecules. Quantitative differences are found with denatured DNA's. The strongest complement fixation is observed with (G+C)-rich denatured DNA. The reaction with denatured DNA is only partially inhibited by DNA digest. These antibodies obviously react with sequential determinants containing bases. Therefore, they are induced by a different mechanism of sensitization.     

Comparison of theoretical denaturation maps of phiX174 and SV40 with their gene maps.

When the theoretical denaturation maps of phiX174 and SV40 are compared with their gene maps, it is observed that the beginning and the end of each gene in these two DNA's fall in a region of lower melting temperatures. Local (A+T)-contents evaluated from the known sequences at these regions support the above implication that the beginnings and the ends of nearly all the genes in phiX174 and SV40 are relatively rich in (A+T)-content.     

Immune response to deoxyribonucleic acid (DNA) of African trypanosomes

Kushimo J. B. and Akinrimisi E. O. Immune response to deoxyribonucleic acid (DNA) of African trypanosomes. International Journal for Parasitology12: 537–540. Rabbits immunized with methylated bovine serum albumin (MBSA) complexes of nuclear DNA from either T. brucei or T. vivax which have low content of Adenine + Thymine (A + T) produced only IgM antibodies to denatured DNA. On the other hand rabbits immunized with MBSA complexes of denatured kinetoplast DNA (K-DNA) from either T. brucei or T. vivax which are rich in A + T content elicit both IgM and IgG antibodies. However the amount of IgG antibodies was higher than IgM antibodies. There seems to be a correlation between % AT content of immunogen and average ratio of maximum precipitable IgG to IgM (IgG)/(IgM).     

Transforming activity of phage T4r+ DNA, treated with ultraviolet light, nitrous acid, hydroxylamine and visible light in the presence of methylene blue]

The efficiency of phages T4rIIB-638v+ and T4rIIB-638v- transformation by native and denatured DNA treated with UV, nitrous acid, hydroxylamine and visible light in the presence of methylene blue is studied. A greater transformation efficiency of UV-irradiated T4r+ phage native and denatured DNA was observed in the v+ recipient as compared with v- recipients. Denatured donor DNA treated with nitrous acid has higher transformation activity in spheroplasts infected with T4v+ phage than in those infected with T4v- phage. Native donor DNA, treated with methylene blue and visible light-irradiated, developed a decrease of the transformation activity in T4v- phage-infected spheroplasts as compared with T4v+ phage-infected spheroplasts. Hydroxylamine treatment of native and denatured donor DNA did not reveal any differences in the transforming activity for v+ and v- recipients. Denatured donor DNA was more resistant to the effect of hydroxylamine than native DNA.     

Antibodies elicited by defined oligodeoxyribonucleotide sequences.

Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.     

Similarity of the structure of DNA from a variety of sources

DNA's from diverse cells of different species and from diverse tissues give the same x-ray diffraction pattern. The presently observable structure of DNA appears, then, to be the same in all cells. Thus, DNA in the resting state-the stored genetic material, from sperm of Paracentrotus lividus, Arbacia lixula, and salmon and from T(2) and T(7) bacteriophage-gives a pattern indistinguishable from DNA from very rapidly dividing cells, e.g., human acute leukemic leukocytes, human leukemic myeloid cells, mouse sarcoma 180, and bacteria-E. coli and pneumococci-during their logarithmic growth. The same x-ray patterns are given by DNA's from more slowly dividing tissues, e.g. calf liver, calf thymus, and human normal and leukemic lymphatic tissue. DNA from chicken erythrocytes-a DNA presumably metabolically inert-gives a similar picture. DNA's from several sources with a wide range in nitrogen base ratios, prepared independently by different workers using various methods, have given final products in varying yield; these all gave the same x-ray pattern, suggesting that all DNA is in the double-helical configuration. Finally, separation of the DNA molecule into a number of fractions with a varying adenine + thymine:guanine + cytosine ratio, but a constant adenine:thymine and guanine:cytosine ratio, each giving the same x-ray pattern as the original whole molecule, suggests that DNA cannot exist in significant amounts in forms other than the double-helix. X-ray diffraction photographs of sperm heads, extracted nucleoprotamine, calf thymus nuclei and extracted nucleohistone, and of chicken erythrocyte nuclei, are not all as well defined as those given by extracted DNA, but it is clear from the general characteristics of the pattern that much of the DNA bound to protein in these nuclei has the usual helical configuration, and that the double-helical structure of DNA exists in the cell and is not an artifact.     

Salt effects on the denaturation of DNA

When DNA's of differing GC:AT base ratios, e.g. synthetic poly dAT, T4 DNA,calf thymus DNA, E. coli DNA, and M. lysodeikticus DNA, are heat-denatured at neutral pH in increasing concentrations of N(a)(2)SO(4) or C(s)(2)SO(4) as supporting electrolytes,the variation of melting temperature with average base composition, dT(m)/dX(G)(C), changes from 45°C (in 0.002M Na) to ll°C (in 4.5M Na) and from 42°C (in 0.002M Cs) to 3°C(in 4.5M Cs). The decrease of dT(m)/dX(G)(C) is a monotonic function of decreasing water activity in the salt solutions. We interpret this decreased composition dependence of the thermal stability of the various DNA's as being due to a destabilization of the GC base pairs relative to the AT base pairs by the concentrated salt media. A simple quantitative treatment shows that k = 8GC/SAT decreases from a value of 4.14 (in 0.01MN(a)) to 1.86 (in 3M Na) and from 4.18 (in 0.01M Cs) to 1.42 (in 3M Cs). SAT is the equilibrium constant for the formation of a hydrogen-bonded AT base pair from a pair of unbonded bases at the junction between a helical region and a denatured region and SGC is the like constant for the formation of a GC base pair. These results corroborate our previous findings of a strongly reduced composition dependence of the negative logarithm of the methylmercuric hydroxide concentration necessary to produce 50% denaturation when the helix-coil transition of DNA is studied in concentrated Cs(s)SO(4)(ultracentrifugation) instead of in dilute N(a)(2)SO(4) (ultraviolet spectrophotometry).     

Sedimentation Rate as a Measure of Molecular Weight of DNA

Zone centrifugation of mixtures of two labeled DNA's at low concentrations in density gradients of sucrose permits accurate measurement of relative sedimentation rates. The individual rates are constant during the run. Measurements with DNA's from phages T2, T5, and lambda conform to the relation D₂/D₁ = (M₂/M₁)^0.35, where D and M refer to distances sedimented and molecular weights of the DNA pair. The results show that high molecular weight DNA's sediment artificially fast in the optical centrifuge, owing to a hitherto unknown effect of molecular interactions. The molecular weight of lambda DNA is 31 million, measured either from sedimentation rate or from tests of fragility under shear.     

Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states.

Commercial 14C-labeled KB cell DNA, widely used to assay sera for anti-DNA antibodies, was chromatographed on benzoylated-naphthoylated-DEAE-cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaC1 (KB fraction I) characteristic of ds-DNA. Fifty-five percent of the label eluted with 50% formamide-1 M NaC1 (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss-DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds-DNA. after pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C-KB-cell DNA preparation was ds-DNA with ss regions which was undetectable by HAP chromatography. 3H-ds-DNA and circular 3H-ss-DNA prepared from T7 and phiX174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non-SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti-ds-DNA antibodies on the basis of KB cell DNA testing and detectable antibodies against KB fraction 1 or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non-SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phiX174 DNA, KB fraction II DNA and alkali-denatured T7 DNA. The data support the conclusions that 1) false positive tests for anti-ds-DNA antibodies can result from contamination of ds-DNA with ds-DNA having ss regions, and 2) non-SLE sera do not contain antibodies specific for ds-DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Influence of antibodies against DNA on the renaturation of DNA.

The treatment of denatured T4 phage DNA with antiserum for the DNA of this phage, containing antibodies against glucosylated 5-hydroxymethylcytosine, decreases the ability of DNA for renaturation. The greatest inhibiting activity is possessed by antiserum for T4 phage DNA irradiated with UV light, which contains antibodies not only against glucosylated 5-hydroxymethylcytosine, but also against the usual nitrogen bases. Antiserum against E. coli DNA, containing antibodies to the usual nitrogen bases, in equal dilutions with the antisera indicated above, shows less inhibitory activity on the renaturation of T4 phage DNA.     

Solid phase immunoenzyme analysis of the immunospecificity of DNA from calf spleen alkylated by thiophosphamide

DNA binding activity of rabbit antiserum against calf spleen DNA's modified by thiophosphamide (DNA-T) was studied by means of solid enzyme immunoassays (ELISA). The studies demonstrated the preferential binding of the immobilized DNA-T compared to immobilized single-stranded DNA (ss-DNA) and only small preference compared to native DNA. Two antisera against DNA-T were purified by affinity chromatography on a ss-DNA-CNBr agarose from antibodies to calf spleen ss-DNA. They interacted only with the immobilized DNA-T, but not with ss-DNA or native DNA. These results demonstrated that DNA modification by thiophosphamide, decreases the immunogenicity of usual nitrogen-containing DNA bases, but detected new immunogenic specificity for adducts. Detection of new immunogenic specificity in DNA's alkylated by thiophosphamide, resulted in the development of a sensitive enzyme immunoassay for the detection of these adducts in nucleic acids, in monitoring their formation, persistence and repair damages in DNA.     

Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base.

Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.     

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNA's and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.     

Ca2+ binding environments on natural and synthetic polymeric DNA's.

Here are reported 43Ca nmr chemical shift and line width measurements obtained during 43CaClO4 titrations of two natural and two synthetic polymeric DNA's. Titrations of the natural DNA's demonstrate the existence of at least two classes of bound 43Ca2+. The 43Ca2+ nmr relaxation and chemical shift behavior observed during titration of C. perfringens DNA (31%GC) is dominated by a delocalized, non-specific interaction. In contrast, titration of M. lysodeikticus DNA (72% GC) indicates that a small fraction of the 43Ca2+ experiences significant motional retardation and/or an increase in the electric field gradient when associated to the DNA, and thus appears to be locally bound to discrete sites on the DNA. These results, and previous results for calf thymus DNA (39% GC) demonstrate that higher GC content correlates with an increase in favorable Ca2+ binding environments. Titrations of synthetic DNA demonstrate that Ca2+ binding is remarkably sensitive to local DNA structure.     

DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

Interaction of metal ions with nucleic acids and related compounds. II. Studies on Au(III)-nucleic acid system

The nature of interaction of Au(III) with nucleic acids was studied by using methods such as uv and ir spectrophotometry, viscometry, pH titrations, and melting-temperature measurements. Au(III) is found to interact slowly with nucleic acids over a period of several hours. The uv spectra of native calf-thymus DNA 9pH 5.6 acetate buffer containing (0.01M NaCIO₄) showed a shift in λ max to high wavelengths and an increase in optical density at 260 nm. There was a fourfold decrease in viscosity (expressed as η_sp/c). The reaction was faster at pH 4.0 and also with denatured DNA (pH 5.6) and whole yeast RNA (pH 5.6). The order of preference of Au(III) (as deduced from the time of completion of reaction) for the nucleic acids in RNA > denatured DNA > DNA. The reaction was found to be completely reversible with respect KCN. Infrared spectra of DNA-Au(III) complexes showed binding to both the phosphate and bases of DNA. The same conclusions were also arrived at by melting-temperature studies of Au(III)-DNA system. pH titrations showed liberation of two hydroxylions at r = 0.12 [r = moles of HAuCl₄ added per mole of DNA-(P)] and one hydrogen ion at r = 0.5. The probable binding sites could be N(1)/N(7) of adenine, N(7) and/or C(6)O of guanine, N(3) of cytosine and N(3) of thymine. DNAs differing in their (G = C)-contents [Clostridium perfingens DNA(G = C, 29%), salmon sperm DNA (G + C, 42%) and Micrococcus lysodeikticus DNA(G + C, 29%), salmon sperm DNA (G = C, 72%)] behaved differently toward Au(III). The hyperchromicity observed for DNAs differing in (G + C)-content and cyanide reversal titrations indicate selectivity toward ( A + T)-rich DNA at lw values of r. Chemical analysis and job's continuous variation studies indicated the existence of possible complexes above and below r = 1. The results indicate that Au(III) ions probably bind to hte phosphate group in the initial stages of the reaction, particularly at low values of r, and participation of the base interaction also increases. Cross-linking of the two strands by Au(III) may take place, but a complete collapse of the doulbe helix is not envisaged. It is probable that tilting of the bases or rotaiton of the bases around the glucosidic bond, resulting in a significant distrotion of the double helix, might take place due to binding of Au(III) to DNA.     

Polynucleotide Homologies of Brucella Deoxyribonucleic Acids

Deoxyribonucleic acids (DNA's) extracted from organisms presently placed in the genus Brucella (B. abortus, B. melitensis, B. neotomae, and B. suis) possessed very similar polynucleotide sequences. Unlabeled, single-stranded DNA fragments from B. abortus, B. melitensis, B. neotomae, and B. suis were equally effective in competing with the interaction of corresponding radiolabeled, single-stranded DNA fragments with their homologous DNA-agars. Unlabeled fragments of B. ovis, however, did not compete as effectively as the homologous, unlabeled DNA's, and this organism, therefore, had a detectably different polynucleotide composition. The mole percentages of guanine plus cytosine in Brucella DNA's (56 to 58%) were also similar. DNA's from Francisella tularensis, Escherichia coli, and the slow loris did not compete.     

Immunochemical studies on the correlation between conformational changes of DNA caused by ultraviolet irradiation and manifestation of antigenicity.

The conformational changes of double-stranded DNA induced during irradiation with ultraviolet, were immunologically investigated. These studies revealed that at least two distinct antigenic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.     

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.