Immunofluorescent staining of mammalian nuclei and chromosomes with a monoclonal antibody to triplex DNA

Triplex DNA is an unusual conformation of DNA formed when two pyrimidine nucleotide strands share a common purine strand. A monoclonal antibody, demonstrated by numerous criteria to be specific for triplex DNA, was used to investigate the presence and distribution of this unique DNA configuration in nuclei and chromosomes of mouse LM cells and human lymphocytes. Indirect immunofluorescence microscopy revealed that constitutive heterochromatin in acetic-methanol fixed mouse nuclei was usually, but not always immunofluorescent, suggesting possible cell cycle related variations in the amount of triplex DNA or its accessibility in this condensed chromatin. In fixed mouse and human chromosomes, there was a positive correlation between immunofluorescent staining patterns, Hoechst 33258 banding, and G- and/or C-banding patterns. Unfixed, isolated mouse chromosomes also reacted positively with the antibody, particularly when they were gently decondensed by exposure to low ionic conditions at neutral pH. This result indicates that fixation is not mandatory for antibody staining, suggesting that some mammalian chromosomal DNA may be naturally organized in a triplex configuration. However, there is a possibility that fixation may facilitate the formation of additional triplex DNA complexes in potential sequences or expose previously inaccessible triplex DNA. The precise correspondence between the immunofluorescent patterns produced by anti-triplex DNA antibodies and G- and C-bands known to represent regions of chromatin condensation, suggests a potential role of triplex DNA in chromosome structure and regional chromatin condensation.     

High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody

A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.     

A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.     

Cross-reaction of a monoclonal anti-filaggrin antibody with glycogen

A mouse monoclonal anti human filaggrin antibody was found to bind keratohyaline granules of normal epidermis as well as of premalignant and malignant lesions in formalin-fixed tissue sections. In addition, an unexpected binding of this antibody with cells containing glycogen and other PAS positive substances was found, which could be abolished by adsorption of the anti-filaggrin antibody with glycogen or pretreatment of the sections with diastase.     

Cross-reaction of a monoclonal anti-filaggrin antibody with glycogen

Summary A mouse monoclonal anti human filaggrin antibody was found to bind keratohyaline granules of normal epidermis as well as of premalignant and malignant lesions in formalin-fixed tissue sections. In addition, an unexpected binding of this antibody with cells containing glycogen and other PAS positive substances was found, which could be abolished by adsorption of the anti-filaggrin antibody with glycogen or pretreatment of the sections with diastase.     

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.     

Chromosomal localization of the ovine beta-casein gene by non-isotopic in situ hybridization and R-banding.

The ovine beta-casein gene (CNS2) has been mapped to a specific chromosome band using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe pTZ-E4 was a fragment of the ovine beta-casein gene inserted in the plasmid pTZ18R and labeled with biotin-11-dUTP. It hybridized to band q32 of ovine chromosome 4. The discrepancy between this result and the previous localization of this gene on cattle chromosome 6 may be explained by the very great similarity of the banding patterns of ovine and bovine chromosomes 4 and 6.     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody

BALB/c mice were repeatedly immunized with a galactosyl transferase- rich microsomal fraction of rat myeloma cells. Spleen cells were subsequently fused with Sp2/0 mouse myeloma cells, the resulting hybridomas were cloned, and their secreted Ig was screened for reactivity with antigens belonging to the Golgi complex. One such monoclonal antibody, 6F4C5, gave especially intense immunofluorescent staining of the Golgi area of myeloma cells and fibroblasts. It recognized two proteins bands on immunoblots of gel-fractionated cell lysates: a major one with an estimated Mr of 54,000 and a minor one at 86,000. Both proteins were concentrated in microsomal fractions isolated at low ionic strength. They were hydrophilic judging from partitioning of a Triton X-114 cell lysate. Both were cytoplasmically oriented as demonstrated by protease and high KCl treatments of postmitochondrial supernatants and microsomal fractions. Neither was retained by columns of insolubilized wheat germ agglutinin or concanavalin A, which suggests that they are not glycoproteins. Their more detailed location in the Golgi complex was studied by immunoelectron microscopy, using a saponin permeabilization procedure and peroxidase-conjugated reagents. The observed staining was restricted to two or three cisternae in the medial part of the stack. Nevertheless, differential centrifugation experiments indicated that the two antigens may be recovered in distinct subcellular fractions: this may be related to the unexpected observation that rather low salt concentrations strip the antigens from microsomal fraction.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.     

Contamination of a monoclonal antibody with LDH-virus causes interferon induction

Interferon induction occurred unexpectedly during an in vivo study using a mouse monoclonal antibody. The interferon was typed alpha/beta and the titer reached a maximum at 24 hours in contrast with other inducers. Similar results were obtained with a virus pool derived from the antibody and with a LDV reference strain. MAP-testing of the monoclonal antibody revealed contamination with lactate dehydrogenase virus (LDV). The production of IFN seems to be controlled genetically. This experimental error demonstrates the importance of an appropriate quality control of biological materials.     

Characterization of a monoclonal antibody reacting with histone H3

A hybridoma cell line, 1GB3, has been obtained from a fusion between SP/O-Ag 14 myeloma cells and lymphocytes from BALB/c mice immunized with rat liver nuclear proteins. This hybridoma secreted a monoclonal antibody of the IgG2b class which reacted specifically with histone H3 in enzyme-linked immunosorbent assay (ELISA) as well as in immunoblotting and immunodot assays. Stringent test conditions were necessary to eliminate the presence of nonspecific or contaminating reactions with other histones than H3. The monoclonal antibody appears to recognize an epitope situated in the N-terminal residues 20-50 of histone H3; it recognizes this epitope in the octamer aggregate of core histones but not in the core particle.