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B D Stollar

Publications and source records attributed to B D Stollar.

At least 109 records · Page 6Linked to original sources
PubMed1984

Z-DNA immunoreactivity of Drosophila polytene chromosomes. Effects of the fixatives 45% acetic acid and 95% ethanol and of DNase I nicking.

Drosophila salivary chromosomes have been isolated at neutral pH and physiological ionic strength. They display only background level binding of antibodies against Z-DNA. Following exposure to the commonly used fixative 45% acetic acid all of the polytene chromosomes, X and autosomes, show a massive increase in anti-Z-DNA antibody binding. The enhancement from background to intense fluorescence occurs whether the chromosomes are stabilised by two orders of magnitude lower concentration of formaldehyde than that used to minimise protein extraction in classical acid squash preparations, or by physiological concentrations of spermine and spermidine. Nicking of acetic acid-treated chromosomes by DNase I dramatically reduces their Z-DNA immunoreactivity. The histones and non-histones extracted by 45% acetic acid from unfixed and formaldehyde-fixed Drosophila chromatin have been analysed. Exposure of isolated salivary chromosomes to the non-protein-extracting fixative 95% ethanol also enhances Z-DNA immunoreactivity. All of these phenomena must be taken into account in the search for the Z-DNA conformation in cells by cytological techniques.

Acetates↗
PubMed1984

Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies.

Monoclonal anti-DNA antibodies produced by hybridomas derived from MRL-lpr/lpr mice and human lupus patients were found to bind to the cytoskeleton of mink lung cells. When tested by indirect immunofluorescence, 17/29 human monoclonal anti-DNA antibodies reacted with the cytoskeleton; 4 of the 29 also produce antinuclear reactions with epithelial cells. The cytoskeletal staining was not inhibited by prior treatment of the cells with DNase, but it was completely blocked by prior incubation of the monoclonal antibodies with DNA and other nucleic acids. The ability of the polynucleotides to inhibit the cytoskeletal staining corresponded to their ability to bind to the antibodies in competitive immunoassays. An (Fab')2 preparation of a monoclonal antibody bound to the cytoskeleton as well as the whole immunoglobulin. The effect of colcemid on the staining pattern, the blocking effect of a monoclonal antivimentin antibody, and results with nitrocellulose blots of cellular proteins indicated that the cytoskeletal protein to which the antibodies bound was vimentin.

Animals↗
PubMed1984

The specificity of antibodies elicited by poly(dG).poly(dC).

Antibodies have been raised to the synthetic DNA polymer poly(dG).poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.

Antibody Specificity↗
PubMed1984

Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.

Actins↗
PubMed1984

In vivo effects of antibodies against a high frequency idiotype of anti-DNA antibodies in MRL mice.

The in vivo effects of a rabbit antiserum against the dominant idiotype of MRL-lpr/lpr anti-DNA antibodies (termed H130) were studied in MRL-lpr/lpr, MRL/++, and BALB/c mice. Under the conditions we examined, the anti-idiotype did not suppress anti-DNA antibodies or the H130 idiotype in either MRL-lpr/lpr or MRL/++ mice. By contrast, MRL/++ and BALB/c mice responded to the antiserum by producing antibodies with the H130 idiotype. The increase in levels of the idiotype was accompanied by a rise in anti-DNA antibodies only when F(ab')2 fragments of the rabbit antiserum were administered. Only about 10% of the induced H130+ immunoglobulins were anti-DNA antibodies. An immunoregulatory disturbance in MRL-lpr/lpr mice may account for their resistance to anti-idiotypic antibodies. Treatment of autoimmune diseases with anti-idiotypes may have the undesired effect of augmenting the production of autoantibodies.

Animals↗
PubMed1984

Responsiveness of autoimmune and normal mice to nucleic acid antigens.

To test the possibility that anti-DNA antibody formation in autoimmune disease is related to an exceptional responsiveness to nucleic acid immunogens, we examined the ability of normal and autoimmune strains of mice to produce antibodies after immunization with DNA and synthetic helical DNA analogues. Autoimmune mice (MRL/++) did not respond significantly to denatured DNA-methylated BSA in adjuvant (in comparison with adjuvant alone). They did, however, respond to helical structures that differed from B-DNA, including poly-(dG) . poly(dC), poly(dT-dG) . poly(dC-dA), and left-handed Z-DNA. Normal mice (C57BL/6) responded to denatured DNA and, after immunization with the other polymers, produced antibodies of equal or higher titer than those of MRL/++ animals. Neither strain responded to native DNA. The induced anti-nucleic acid antibodies were highly selective and reacted only with the immunogen. In contrast, both lupus autoantibodies and antisera from normal animals that received adjuvant alone cross-reacted with multiple nucleic acid antigens. This result, and the finding that MRL/++ mice were poor responders to nucleic acid immunogens, suggest that different clones and pathways are involved in nucleic acid-induced and spontaneous anti-DNA antibody formation.

Animals↗
PubMed1983

Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II.

Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme.

Animals↗
PubMed1983

Mapping of histone H5 sites on nucleosomes using immunoelectron microscopy.

The location of histone H5 on nucleosomes has been determined by binding anti-H5 antibodies to dinucleosomes, and recording the position of the bound IgG molecules using electron microscopy. Two types of antibody were employed, a total IgG fraction prepared from rabbits immunized with H5/RNA, which reacted to all three domains (NH2-terminal, central globular, and COOH-terminal) of H5, and an immunospecific subfraction which bound only to the central globular peptide. After reacting with dinucleosomes, both types of antibody were localized primarily in the linker DNA entry/exit region, but the whole antibody showed a much greater affinity for the linker DNA itself than did the antiglobular peptide antibody. These results provide direct support for the concept that H5, and by inference H1, is located at the linker DNA entry/exit site of the nucleosome, and further suggest that it is the central globular portion of the molecule that is most closely associated with this site. An interaction of one or both termini of H5 with the linker DNA is also indicated.

Animals↗
PubMed1983

Idiotypic cross-reactions of monoclonal human lupus autoantibodies.

Idiotypic cross-reactions were evaluated in 60 polynucleotide-binding monoclonal lupus autoantibodies produced by human-human hybridomas that were derived from seven unrelated patients with SLE. Three antiidiotype reagents were prepared by immunization of rabbits or a mouse with monoclonal autoantibodies from two patients. Binding of the three reagents to their corresponding idiotypes was inhibited by one or more polynucleotides, an indication that the antiidiotypes reacted with the variable regions of the autoantibodies. Each antiidiotype appeared to detect a different idiotypic determinant. Of the 60 monoclonal autoantibodies tested, 40 reacted in one or more competitive immunoassays; 15 reacted with one antiidiotype, 10 reacted with two antiidiotypes and 15 reacted with three antiidiotypes. A monoclonal antiidiotype reagent cross-reacted with autoantibodies from six of the seven patients. The idiotypic cross-reactions of immunoglobulins from unrelated patients suggest that the autoantibodies are derived from related families of germ line genes that are expressed by patients with SLE.

Animals↗
PubMed1983

Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas.

We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

Adult↗
PubMed1983

Antibodies to calf thymus RNA polymerase II from egg yolks of immunized hens.

Polyclonal antibodies to calf thymus RNA polymerase II were raised in laying hens. Up to 75 mg of immunoglobulin/egg yolk were extracted by the polyethylene glycol procedure of Roeder (Roeder, R.G. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds) pp. 285-330, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The concentration of specific antibody in egg yolks (IgY) was comparable to that of serum as measured by enzyme-linked immunoassay. Purified antibody was shown to be directed against enzyme by removal of enzyme activity in immune complexes precipitated by rabbit anti-chicken IgY. The antibodies recognized several of the subunits of the enzyme as determined by their reactivity with polypeptides transferred to nitrocellulose paper after gradient sodium dodecyl sulfate-gel electrophoresis. Production of antibodies in laying hens may facilitate the study of other highly conserved antigens that are poorly immunogenic in mammalian hosts.

Animals↗
PubMed1983

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus.

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Animals↗
PubMed1983

Lack of Z-DNA conformation in mitomycin-modified polynucleotides having inverted circular dichroism.

Poly(dG-dC) . poly(dG-dC) and Micrococcus lysodeikticus DNA were modified by exposure to reductively activated mitomycin C, an antitumor antibiotic. The resulting covalent drug-polynucleotide complexes displayed varying degrees of CD inversions, which are strikingly similar to the inverted spectrum observed with Z-DNA. The following criteria have been used to establish, however, that the inverted CD pattern seen in mitomycin C-polynucleotide complexes does not reflect a Z-DNA conformation. (i) The ethanol-induced transition of poly(dG-dC) . poly(dG-dC) from B to Z conformation is not facilitated but rather is inhibited by mitomycin C modification. This may be due to the presence of crosslinks, (ii) Radioimmunoassay indicated no competition for Z-DNA-specific antibody by any of the mitomycin C-modified polynucleotides, (iii) 31P NMR of the complexes yielded a single relatively narrow resonance, which is inconsistent with the dinucleotide repeat characteristic of Z-DNA. Alternative explanations for the inverted CD pattern include a drug-induced left-handed but non-Z conformational change or the superposition of an induced CD onto the CD of B-DNA due to drug-base electronic interactions. These results illustrate the need for caution in interpreting CD changes alone as an indication of Z-DNA conformation.

Circular Dichroism↗
PubMed1983

Normal mice express idiotypes related to autoantibody idiotypes of lupus mice.

Spleen and fetal liver B cells of normal mice synthesized idiotypes shared by anti-DNA autoantibodies of genetically autoimmune mice. Some of the idiotypes were specific for DNA; the majority, however, were not. The findings indicate that the autoantibody idiotypes are related to a conserved family of antibody variable regions that are present in normal animals.

Animals↗
PubMed1983

Z-DNA and the polytene chromosome.

Indirect immunofluorescence shows that antibodies specific for the Z conformation of DNA bind to D. melanogaster polytene chromosomes in a specific and reproducible pattern. In our acetic-acid-fixed preparations, the binding is predominantly to interband regions, to puffs, and to some specific bands. As might be expected, the pattern of binding can be affected by cytological procedures. Bands and interbands show significant differences in their susceptibility to these procedures. Although further study is needed, our studies give no evidence that acetic acid treatment of intact cells is capable of causing conversion of B-form and Z-form DNAs. Instead, the experiments suggest that the Z conformation is preferentially available in interbands and a few other chromosome regions.

Animals↗