Specificity of anti-nucleoside antibodies (Short Communication)

The method of conjugation of a nucleoside or related compound to a carrier protein may have a significant effect on the specificity of the antibodies elicited. It is demonstrated, by means of the membrane-filtration assay, that anti-isopentenyladenosine antibodies produced by the ;periodate procedure' are much more reactive with the periodate-oxidized form of the nucleoside than with the parent compound. In addition, the simplicity and specificity of the assay used suggests its use as a sensitive radioimmunoassay for this multifunctional nucleoside.     

Probing the nematode surface

The surface of parasitic nematodes has been well studied with respect to its structural and immunological properties, but little is known about its biophysical nature and the role this plays in the host-parasite relationship. In this article, Clare Roberts and Jay Modha highlight some biophysical features of nematode surfaces and discuss their recent findings regarding mechanisms controlling surface-associated biophysical phenomena observed in parasitic nematodes during infection or culture in medium simulating the mammalian host environment. The nematode surface is distinct from the plasma membrane, nevertheless some parallel features exist and are described.     

Probing myosin head structure with monoclonal antibodies

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.     

Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies.

We have probed the structures of monomeric and oligomeric gp120 glycoproteins from the LAI isolate of human immunodeficiency virus type 1 (HIV-1) with a panel of monoclonal antibodies (MAbs); most of these MAbs are directed against continuous epitopes. On native monomeric gp120, most of the first conserved (C1) domain is accessible to MAbs, although some regions of C1 are relatively inaccessible. All of the MAbs directed against the C2, C3, and C5 domains bind preferentially to denatured monomeric gp120, indicating that these regions of gp120 are poorly accessible on the native monomer, although the extreme C terminus in C5 is well exposed. Segments of the V1, V2, and V3 loops are exposed on the surface of monomeric gp120, although the base of the V3 loop is inaccessible. A portion of C4 is also available for MAb binding on monomeric gp120, as is the extreme C terminus in C5. However, on oligomeric gp120-gp41 complexes, only the V2 and V3 loops (and perhaps V1) are well exposed and a segment of the C4 region is partially exposed; continuous epitopes in C1 and C5 that are accessible to antibodies on monomeric gp120 are occluded on the oligomer. Although deletion of the V1, V2, and V3 loops resulted in increased exposure of several discontinuous epitopes overlapping the CD4-binding site, the exposure of most continuous epitopes on the monomeric gp120 glycoprotein was not affected. These results imply a HIV-1 gp120 structure in which the conserved continuous determinants are inaccessible; in some cases, this inaccessibility is due to intramolecular interactions between conserved regions, and in other cases, it is due to intermolecular interactions with other components of the glycoprotein spike. These findings have implications for the design of subunit vaccines based on gp120.     

Probing DNA polymerase alpha with monoclonal antibodies

DNA polymerase alpha was purified from human KB cells by immunoaffinity chromatography. Enzyme activity was inhibited by three different monoclonal antibodies (SJK-132, SJK-211, SJK-287). Kinetic analysis showed that each antibody neutralized polymerase activity by a different mechanism. SJK-132 was competitive with DNA indicating it interacts with the DNA binding domain of the polymerase. SJK-287 showed a biphasic response to dCTP suggesting two dCTP binding sites exist on polymerase alpha. SJK-211 was non-competitive with DNA, dCTP and dATP.     

Binding of cis- and trans-diamminedichloroplatinum(II) to deoxyribonucleic acid exposes nucleosides as measured immunochemically with anti-nucleoside antibodies

We report the use of anti-nucleoside antibodies to probe for local denaturation of calf thymus DNA upon binding of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, and the biologically inactive analogues trans-diamminedichloroplatinum(II), trans-DDP, and chloro(diethylenetriamine)platinum(II) chloride, [Pt(dien)Cl]Cl. These antibodies specifically recognize each of the four DNA nucleosides. They bind well to denatured DNA, but not to native DNA in which the bases are less accessible owing to Watson-Crick duplex structure. At relatively high levels of modification (D/N approximately 0.1), cis-DDP causes significant disruption of DNA base pairing as reflected by the increased binding of anti-cytidine, anti-adenosine, and anti-thymidine antibodies. At lower levels of platinum adduct formation, however, all four anti-nucleoside antibodies bind more to DNA modified with trans-DDP. This result indicates that adducts formed by trans-DDP disrupt the DNA structure to a greater extent than those formed by cis-DDP at low D/N ratios. Modification of DNA by the monofunctional complex [Pt(dien)Cl]Cl does not affect its recognition by anti-nucleoside antibodies, demonstrating that base pair disruption is a consequence of bifunctional binding. The relative anti-nucleoside antibody recognition of cis-DDP-modified DNA is anti-cytosine greater than anti-adenosine approximately anti-thymidine much greater than anti-guanosine, consistent with the major adduct being an intrastrand d(GpG) cross-link. These results reveal that base pair disruption in a naturally occurring DNA modified by either cis-DDP or trans-DDP is sufficient to be detected by protein (antibody) binding. The relevance of these findings to current ideas about the molecular mechanism of action of cis-DDP is discussed.     

Probing eukaryotic RNA polymerases B with monoclonal antibodies

Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora comata were selected on the basis of different subunits recognition and inhibitory effect on enzyme activity. A library of 10 antibodies biased toward B180, B145, B39, B23,5 and B11 subunits was constructed. Most of these antibodies also recognize yeast, wheat germ and calf thymus RNA polymerase B. Subunits bearing antigenic determinants are not always homologous in Podospora and yeast enzyme. As some of these antibodies strongly inhibit enzyme activity they constitute potent probes for functional studies of corresponding subunits.     

Characterization of anti-Z-RNA polyclonal antibodies: epitope properties and recognition of Z-DNA

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

Binding of anti-nucleoside antibodies reveals different classes of DNA in the chromosomes of the kangaroo rat (Dipodomys ordii)

The binding of highly purified anti-nucleoside antibodies to fixed metaphase chromosomes of the kangaroo rat (Dipodomys ordii) revealed the presence of different classes of DNA in different regions of the chromosomes. To permit antibody binding, the chromosomal DNA was first made single-stranded by either ultraviolet irradiation, which denatures some classes of AT-rich DNA, or photo-oxidation, which denatures GC-rich DNA. The antibody binding patterns obtained matched the location of the different classes of satellite DNA in kangaroo rat chromosomes. After either denaturation method, anti-5-methylcytidine (anti-M) bound intensely only to the centromeric heterochromatic regions which are known to contain the GC rich, highly methylated HS-β satellite DNA of this species. The basic repeating unit of the HS-β sequence is 5′-ACACAGCGGG-3′ 3′-TGTGTCGCCC-5′ [4]. The binding of anti-M after UV irradiation is permitted by the production of pyrmidine (CC and TC) dimers in the right-hand portion of this repeating sequence, supporting the idea that the 5-methylcytosine residues are in this portion. After photo-oxidation, anti-cytidine (anti-C) and anti-adenosine (anti-A) also showed intense binding to the centromeric heterochromatin. In addition, these antibodies showed moderately intense binding to non-centromeric heterochromatic regions, which contain the relatively GC-rich HS-α and MS satellite DNAs. After UV irradiation, anti-A binding produced a banding pattern identical to the quinacrine (Q-band) pattern, with bright chromosome arms and very dull centromeric heterochromatic regions, while anti-C showed moderate binding in the centromeric regions and fairly even but weak binding elsewhere.The results have clarified the way in which anti-nucleoside antibodies react with chromosomal DNA. The reactivity of anti-A, anti-C and anti-M with the partially denatured HS-β satellite DNA supports the idea that antibody binding requires denaturation of a sequence perhaps no more than 5 base pairs long. In addition, it appears that it is not necessary to have more than one identical base in a row to permit antibody binding.     

Chemical footprinting of the interaction between left-handed Z-DNA and anti-Z-DNA antibodies by diethylpyrocarbonate carbethoxylation

Diethylpyrocarbonate (DEPC) carbethoxylates Z-DNA to an increased extent because the reactive N-7 atoms of purine residues appear structurally more accessible on Z-DNA as opposed to B-DNA. This chemical probe was used in DEPC footprinting experiments, which confirm the specificity of binding of anti-Z-DNA monoclonal antibodies and which probe regions of close contact in this DNA-protein complex. Antibody binding to segments of Z-DNA existing in supercoiled plasmids resulted in specific protection from DEPC hyper-reactivity within the Z-DNA segment and induction of hyper-reactivity in purines lying adjacent to the Z-segment. Two different monoclonal immunoglobulin preparations, Z22 and Z44, are shown to generate specific and distinct footprint patterns when bound to the Z-helix. Binding of these antibodies was also found to affect DNA conformation within the Z-DNA segment by influencing the equilibrium between the B- and Z-helical conformations.     

Mapping Z-DNA tracts in plasmid DNA using electron microscopy and gold-labeled antibodies

Using alternating poly(dG-dC).poly(dG-dC) and electron microscopy (EM), a method has been developed for detecting regions of Z conformation in DNA preparations. The procedure was developed with poly(dG-dC).poly(dG-dC) which had been converted to the Z conformation with MnCl2 and mild heat treatment. Conditions were found for reaction of this DNA with polyclonal anti-Z antibodies from rabbit, and further reaction of this mixture with gold-labelled anti-rabbit antibodies from mouse. Spreading of these samples onto air-water interfaces and examination by EM revealed gold particles aligned along strands of poly(dG-dC).poly(dG-dC). The method was refined and simplified using monoclonal antibodies and tested with the 2.2-kb plasmid, pDHg16, carrying a single tract of alternating d(G-C)23. Treatment with MnCl2 and mild heat was not necessary, as the superhelicity of this molecule ensured that the d(G-C) tract was in the Z conformation. Conditions were found for successful conjugation of mouse monoclonal anti-Z antibodies with colloidal gold (G10), 10.7-nm average diameter. The conjugate was then reacted with superhelical pDHg16, stabilized in polyethylene glycol and cross-linked with glutaraldehyde. Examination by EM showed gold particles at one site on the negatively superhelical circular DNA molecule. When these molecules were linearized with PstI, gold particles were found to occur at an average position 35% +/- 3% from one end. This location agrees well with the known position of the center of the alternating d(G-C) tract with respect to the PstI restriction site (36.8%).     

In situ hybridization of an acetylaminofluorene-modified probe recognized by Z-DNA antibodies in vitro

An in situ hybridization procedure, based on the chemical modification of DNA by acetylaminofluorene (AAF), followed by a specific immunoreaction was used to localize a Z-DNA sequence isolated from the satellite DNA of Cebus appella. The AAF probe is localized on the R-band-positive heterochromatic segments of Cebus chromosomes, which strongly react with Z-DNA antibodies. The use of a nonradioactive single-stranded labeled probe confirms the reliability and the rapidity of immunochemical methods for the detection of DNA sequences on chromosomes.     

Analysis of the murine sperm surface with monoclonal antibodies

Hybridomas secreting monoclonal antibodies reactive with murine spermatozoa were produced by fusion of myeloma cells with spleen cells from C57BL/6J mice immunized with spermatozoa from mice of the same strain. All antisperm antibodies were of the mu (mu) immunoglobulin heavy chain class; only one (MS-1) bound S. aureus protein A. Antibody MS-1 recognized an antigen present on the sperm acrosomal cap, on the surface of cells from liver and kidney and from some cultured cell lines. The subunit molecular weight (69000) of the polypeptide reactive with MS-1 was determined by SDS-PAGE analysis of sperm membrane proteins followed by their electrophoretic transfer to nitrocellulose.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.