The Rna Binding Subset of Adenosine Antibodies

Hapten specific antibodies were elicited against adenosine-BSA conjugate (Ado*-BSA) in rats. By radiohapten assay, two major populations of hapten specific antibodies were identified. They were directed against either adenosine trialcohol(A^ox-red) or morpholino-adenosine(Morph-Ade). The association constant (Ka) for the ³H-A^ox-red binding population was determined to be 1.02 × 10⁷ M^-1. Majority of the A^ox-red specific antibodies were crossreactive with Morph-Ade, but they did not cross-react at all with adenosine(A) or deoxyadenosine(dA). ³H-A binding subsets were actually Morph-Ade specific and constitute less than 10% of the hapten specific antibodies. The Ka value for the ³H-A binding was determined to be 4.5 × 10⁶ M^-1. A very minor and highly cross-reactive subset of Morph-Ade specific antibodies participate in RNA binding. Though a larger proportion of Morph-Ade specific antibodies interact with ³H-A, only a fraction of them bind to RNA. The extreme crossreactivity and hence, the highly adaptive nature of the binding sites of the antibodies interacting with RNA, might be a stringent requirement for recognising adenine residues of nucleic acids in solution.     

The Production of High Affinity Monoclonal Antibodies to Human Chorionic Gonadotropin and Their Application to Immunoradiometric Assay

Abstract

Production of monoclonal antibodies against hCG has been studied using hCG as the antigen. This study reports the successful isolation of hybrid clones secreting monoclonal antibodies specific for hCG with an affinity constant higher than 10¹⁰M^?l. Of 23 fusions, only 17 fusions have produced positive clones which secrete antibodies giving high levels of binding with ^l25I-labelled hCG in the supernatant. Finally, 6 different monoclonal antibodies have been isolated; 4 of them, specific for the β-subunit, with a Ka approximately 1.1–4.0 ± 10¹¹M^?1 and 2 others, specific for the α-subunit, presenting an affinity of 2.5 ± 10¹⁰M^?l. When the antibodies specific for the β-subunit are used, specific and highly sensitive radioim-munoassays are obtained after only 3 hrs of incubation. Using iodinated monoclonal antibodies specific for the α-subunit and tubes coated with antibodies against the β-subunit, we have developped sensitive immunoradiometric assays.     

Antigen valence and Fc-localized secondary forces in antibody precipitation

A tetravalent dinitrophenyl hapten, 1,6,11,16-tetra-(?-N-Dnp)-l-lysine₁₆3 was synthesized. Upon interaction with specific antibody of moderate affinity (K_t = 6 × 10⁶ M^?1), a maximum of 80% precipitation occurred. Low pH or 2.5 M guanidine hydrochloride completely inhibited precipitation without significantly affecting hapten binding. Anti-Dnp (Fab')₂ fragments also formed precipitates when mixed with the tetravalent hapten, but these were not soluble in 2.5 M guanidine hydrochloride, and the precipitation itself occurred over a much narrower range of hapten/antibody ratios than observed with intact molecules. These results suggest that a site located in the Fc region assists aggregation and preciptation in intact antibody molecules.     

First order dissociation rates between a subpopulation of high affinity rabbit anti-fluorescyl IgG antibody and homologous ligand

The first order dissociation rates of liganded subpopulations of purified hyperimmune rabbit antifluorescyl IgG antibodies were determined using the method of Green (1963). The dissociation of radiolabeled fluorescyl ligands was measured in the presence of excess unlabeled homologous hapten. Results with three different antibody preparations indicated dissociation rates of 1.3 × 10[su?3, 2.4 × 10^?4 and 2.2 × 10^?6 sec^?1 for the liganded subpopulations. Assuming an association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971), average equilibrium constants ranging from 3.1 × 10¹¹M^?1 to 2.1 × 10¹⁴M^?1 were calculated. These values are among the highest reported for antibody-hapten interactions.     

Cytotoxic effect of ricin A-chain conjugates containing monoclonal antibodies against human erythroid cells

Selective elimination of human erythroblastoid cells by the conjugate of the A-chain of a plant toxin ricin (R_A) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) has been demonstrated. In comparative experiments, MAb HAE3 (IgM) against human glycophorin-A was used. On average, the conjugates obtained contained two A-chain molecules and one antibody molecule. Efficiency of cytotoxic action of native ricin and conjugates was compared both with the amount of binding sites on the surface of K562 cells and the internalization rate of these proteins. The association constants of the proteins proved to be almost the same (k_a = 10⁸ M⁻¹). The ID₅₀ values were 1.1 × 10⁻¹¹, 3.2 × 10⁻¹⁰ and 3.1 × 10⁻⁹ M for ricin, HAE9/R_A and HAE3/R_A, respectively. Ammonium chloride at a concentration of 10 mM increases the cytotoxic effect of the HAE9/R_A conjugate approximately 10 times and does not change the activity of the HAE3/R_A conjugate.     

Structural requirements for recognition of vasopressin by antibody; Thermodynamic and kinetic characteristics of the interaction

The thermodynamic and kinetic properties of two conventional antivasopressin antisera were studied. Values for binding affinity were determined by equilibrium measurements to be Kd = 1.4 and 2.7 × 10^?12M. The kinetic parameters were independently determined. The association rate constants (kas) were calculated by a pseudo-first-order analysis of binding kinetics (ka = 3.1 and 1.7 × 10⁷M^?1 sec^?1). The dissociation rate constants (kds) were measured by dissociating antibody-labelled antigen complexes with large excess of unlabelled antigen (kd = 1.6 and 1.7 × 10^?5 sec^?1). A fairly close agreement was achieved between equilibrium and kinetic evaluation of the affinity.The heterogeneity cannot be assessed through equilibrium experiments because of the very low concentrations of reagents to be handled. However, kinetic studies strongly suggested that the molecular heterogeneity with respect to affinity of the antisera is restricted to a narrow range (5 × 10^?13M to 7 × 10^?12M).Despite their very similar physicochemical properties these two antisera exhibited different fine specificities: the study of cross-reactivities with various analogues of the original hapten showed that one antiserum—5—is clearly directed against the C-terminal moiety of the molecule. The antigenic determinant is a sequential one and composed of the last four aminoacids Cys-Pro-Arg-GlyNH₂, while the other antiserum is not so sensitive to modifications of the last residue GlyNH₂.     

The Production and Characterization of Novel Heavy-Chain Antibodies Against the Tandem Repeat Region of MUC1 Mucin

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH₁). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. K_a values of specific polyclonal anti-peptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 × 10¹⁰ M^{? 1} and 1.4 × 10¹⁰ M^{? 1} respectively.     

Characterization of high-affinity antifluorescyl IgM antibodies

High-affinity IgM rabbit antibodies were elicited using the fluorescein hapten system. Purity and identification of IgM and IgG anti-fluorescyl antibodies was determined by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Ultracentrifugation studies verified that liganded antibodies were of a high mol. wt (901 kd).Comparative analyses of IgM and IgG anti-fluorescyl antibody active sites substantiated the observation that purified IgM preparations, similar to their IgG counterparts, possessed high-affinity antibody active sites. Dissociation rate data confirmed that some IgM molecules within the purified antibody populations possessed association constants at 10¹¹M^?1, comparable with values of 10¹¹M^?1 or greater obtained for some anti-fluorescein IgG populations studied in this laboratory. A diffusion-controlled association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971) was assumed in dissociation rate calculations. This is the first report of purified IgM antibodies possessing exceptionally high affinities.     

The effect of light chain gene expression on the inheritance of an idiotype associated with primary anti-(4-hydroxy-3-nitrophenyl)acetyl(NP) antibodies.

Primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies from C57BL/6 mice were idiotypically defined by a rabbit anti-idiotypic antiserum. The idiotypic marker detected by this antiserum (the NP^b idiotype) requires for its expression a specific heavy (H) chain-lambda (Λ) light (L) chain combination as shown in chain reassociation experiments. The idiotypic binding reaction is inhibitable by hapten. BALB/c and CBA mice also produce large amounts of Λ-bearing anti-NP antibodies, but these do not express the NP^b idiotype as defined by the rabbit antiserum. Since the NP^b idiotype could be reconstituted by reassociating C57 BL/6 anti-NP antibody H chains with either HOPC 1, or BALB/c or CBA anti-NP antibody Λ chains, the absence of the NP^b idiotype in BALB/c and CBA antibodies implies that these animals express different sets of H chain variable (V) regions in the anti-NP response. SJL mice, in contrast to other mouse strains with the Ig-1^b allotype, express only traces of NP^b idiotype in their anti-NP antibodies, the L chains of which are almost exclusively of the χ type. Knowing that SJL mice possess a regulatory gene responsible for the low level of Λ (Λ¹⁰) chains in their immunoglobulins, we have analyzed the progeny of (SJL × BALB/C)F₁, (SJL × BALB/C)F₁ × BALB/C and (SJL × BALB/C)F₁ × SJL for the expression of Λ-bearing anti-NP antibodies and NP^b idiotype. In accord with previous work (SJL × BALB/c)F₁ mice produced substantial amounts of anti-NP antibodies with the NP^b idiotype. Backcross of these mice to BALB/c revealed linkage of the NP^b idiotype to the Ig-1^b allotype, in agreement with previous data. Backcross of the F₁ animals to SJL demonstrated control of the NP^b idiotype by the gene regulating Λ chain expression in serum immunoglobulin. Chain reassociation experiments showed that in SJL mice, the regulation against Λ chain expression prevents the selection in the anti-NP response of those H chains that together with Λ chains constitute the NP^b idiotype. Apparently, these H chains are unable to contribute to a binding site with specificity for the NP hapten when combined with χ chains.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

Autoantibodies against oxidized low density lipoproteins (oxLDL): characterization of antibody isotype,subclass, affinity and effect on the macrophage uptake of oxLDL

Circulating antibodies against oxLDL are present in several inflammatory and autoimmune conditions. Such antibodies are also present in patients with atherosclerosis, although the pathogenic significance of the antibodies is still not known. We have characterized the antibodies with regard to isotype, subclass, affinity and effect on macrophage uptake of oxLDL. Antibodies of IgG and IgM isotype were most common and found both in patients with atherosclerosis and in normal individuals. The subclass of IgG antibodies was mainly IgG2 and IgG3. Scatchard analyses of IgG and IgM antibodies showed that IgG antibodies were heterogeneous with regard to affinity, whereas only one population of high-affinity antibodies was found in the IgM antibody population. The high-affinity populations had an average equilibrium constant (KO) of 8.84 × 10⁹ M^?1 for IgG antibodies and of 1.65 × 10⁹ M^?1 for IgM antibodies. Incubation of ¹²⁵I-oxLDL with purified IgG and IgM from sera with high amounts of antibodies enhanced the uptake of ¹²⁵I-oxLDL in the monocyte-like U937 cell line. Antibody preparations from sera containing no anti-oxLDL antibodies and from sera with antibodies against LDL had less effect on this uptake. The increased uptake was competitively decreased by adding unlabelled oxLDL. This study shows that antibodies against oxLDL are mainly IgG2. IgG3 and IgM. Both IgG and IgM antibodies have a high affinity for the antigen and increase the uptake of oxLDL in a monocyte-like ceil line.     

Base-Specific Binding of Adenosine Antibodies to Double-Stranded DNA

Antibodies raised against adenosine have been reported to react with single-stranded DNA but not with double-stranded DNA. Using a highly sensitive avidin-biotin microELISA we report that these antibodies also react with double-stranded DNA. The binding was specific as it was completely inhibited by the homologous hapten. The results indicate that the antibody populations binding to ssDNA and dsDNA are not cross-reactive. The antibodies were shown to react with the topoisomers of plasmid DNA as assessed by gel retardation assay. The antibodies showed differential binding to restriction fragments of DNA indicating that some of the A residues in dsDNA are accessible to the antibodies.     

Genetic characterization of plasmid formation by N- mutants of bacteriophage lambda.

Cells of plants infected with any of three strains of tobacco mosaic virus (the cowpea, common, and a wheat strain) contain, in addition to full-length virus rods, a heterodisperse population of rod fragments. RNA extracted from purified virus showed that some of these rods were of discrete sizes; the RNAs ranged in molecular weight from about 0.28 to 1.7 × 10⁶, containing 3′-ends identical to the original 3′-end of viral RNA, as judged by their capacities to bind a specific amino acid. Only the cowpea strain produced a rod containing a small (0.28 × 10⁶M_r) RNA, which is known to be the mRNA for coat protein. Each strain produced an RNA of 0.68 × 10⁶M_r (intermediate-length RNA) which coded in vitro (using a wheat germ system) for an ～30,000 M_r polypeptide. Other RNAs (0.9 to 1.7 × 10⁶M_r) were less similar in molecular weights among the three strains, but the predominant in vitro product of each was also the ～30,000 M_r polypeptide. peptide maps comparing the translation products of the short and intermediate RNAs of the cowpea strain showed they were distinct polypeptides. We conclude that at least some of the less-than-full-length viral rods found in preparations of the common and wheat strains of TMV, as well as those previously reported for the cowpea strain, represent the encapsidation of subgenomic portions of the viral RNA which are engendered during viral replication.     

The binding of soluble immune complexes of guinea pig IgG2 to homologous peritoneal macrophages Determination of the avidity constants at 4 °C.

Soluble immune complexes of guinea pig IgG₂ anti-DNP (2,4-dinitrophenyl) antibodies and DNP₁₉BSA (bovine serum albumin) were prepared at a variety of antibody-to-antigen ratios. The mean sizes of the complexes were determined by gel filtration, and equilibrium binding of the complexes to homologous peritoneal macrophages was measured at 4°C. Scatchard plots of the binding data were linear indicating that the complexes were binding to a uniform population of functionally independent membrane receptors. The avidity constants for complex binding to macrophages increased from 15.4 × 10⁷ M^?1 for complexes containing an average of two antibody molecules to 59.0 × 10⁷ M^?1 for complexes containing an average of four, compared with an association constant for monomeric IgG₂ of 0.21 × 10⁷ M^?1. Calculation of the molar-free energy changes accompanying monomer and immune complex binding revealed not only that the intrinsic binding activity of mononer was sufficient to account for the complex binding activities by simple cooperation of linked antibodies, but also that the cross- linking of antibodies with antigen imposed a strain on the antibody-receptor interaction, resulting in complex binding energies that were lower than the values expected from a simple cooperative mechanism. Complex inhibition by monomeric IgG₂ of unrelated antibody specificity was studied, and it was observed that high concentrations of monomer led to an increase in macrophage discrimination between complexes of different size. The relevance of this finding to the in vivo clearance of circulating complexes is discussed.     

N-bromosuccinimide oxidation and amino acid content of isoelectrofocused fractions from a DNP antibody population

The reagent N-bromosuccinimide (NBS) has been employed to investigate the role of antibody tryptophan in the binding of 2,4-dinitrophenyl (DNP) ligands by isoelectric anti-DNP antibody components. NBS titration in 0.1 M acetate—8 M urea (pH 4) buffer established a direct relationship between the extinction coefficients, fluorescence yields, maximum quench values, and tryptophan contents of the isoelectric antibodies. However, there was no consistent correlation between these properties and the relative affinities of these antibodies. Treatment with NBS in 0.05 M acetate (pH 4) buffer resulted in the modification of approximately 50–62 per cent of the tryptophans which were modified in the 0.1 M acetate—8 M urea (pH 4) buffer. This was accompanied by an approximate 15 to 20-fold reduction in the hapten binding affinity of the isoelectric antibodies. The presence of hapten protected from approximately five to nine tryptophans against NBS modification, and prevented to a great extent the reduction in binding activity caused by NBS treatment. Though NBS treatment in the 0.05 M acetate (pH 4) buffer eliminated 80–90 per cent of antibody fluorescence, the residual fluorescence was to some degree quenchable by hapten. This finding prohibits definitive conclusions with regard to the functional role of antibody tryptophan in antigen binding. It is suggested, however, that while tryptophan is instrumental in the maintenance of full hapten binding affinity, its primary influence is environmental (possibly by establishing a hydrophobic region conducive to the binding of DNP ligands) rather than as a contact residue.The amino acid contents of the isoelectric components were found to differ significantly in a number of amino acids. This supports the conclusion that anti-DNP antibodies exhibit a great degree of degeneracy with respect to properties characteristic of the whole antibody molecule.     

Characterization of monoclonal antibodies against prostaglandin E2: Fine specificity and neutralization of biological effects

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E₂ (PGE₂) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB₂, a transformation product of PGE₂. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE₂. Cellular hybridizations were performed and five anti-PGE₂ monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the ω-chain of PGE₂ but their specificity toward the α-chain is more limited. The association constants are greater than to 1 × 10⁹M^?1. The monoclonal antibody 8E.57.71 (K_a = 1.3 × 10¹⁰M^?1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE₂ monoclonal antibodies were found to neutralize the specific binding of [³H]PGE₂ to rat brain hypothalamic receptors and to inhibit the PGE₂ induction of rat fundus muscular contraction.     

Immunoassay development for permethrin residues

Polyclonal antisera were produced in rabbits against two different synthetic immunogens, one of which incorporated 3‐phenoxybenzoic acid (PBA) while the other contained dichlorovinyl cyclopropane carboxylate (CPA). The immunogens were constructed such that the hapten was coupled to the carrier protein through a peptide bond to a six carbon spacing group (6‐amino hexanoic acid, 6‐AHA). Both the anti‐PBA and anti‐CPA antisera obtained were able to detect permethrin when used in an indirect competitive enzyme‐linked immunosorbent assay (IC‐ELISA) format. The detection limits typically obtained with both antisera were 10 mg l^‐1with 50% inhibition of antibody binding (ho) at 100mg l^‐1. Cross‐reactivity with the pyrethroids cyfluthrin, phenothrin and deltamethrin was observed for both antisera, the degree of which was related to the structural similarity of the compound to the immunizing hapten. Further development of the immunoassay for permethrin was examined through use of the anti‐PBA antiserum. Assay performance was improved by negative immunoaffinity chromatog‐raphy of the anti‐PBA antiserum, in which antibodies directed against the six carbon spacing group were removed. In conjunction with an avidin‐biotin amplification step, typical detection limits were 1 mg l^‐1 with an I₅₀ value of 15 mg l^‐1. Assay performance was considerably enhanced by use of a microtitre plate coating antigen which possessed a four carbon spacing group between the hapten and carrier protein. The hapten was also coupled to the spacing group through an ester bond. Typical detection limits for permethrin were 0.5μg l^‐1, with an I₅₀ value of 1 mg l^‐1. This assay was also unaffected by the inclusion of methanol at concentrations of up to 10% by volume. The study indicated the potential usefulness of antibodies raised against compounds which mimic moieties present within larger hapten molecules (anti‐hapten mimic antibodies), particularly where the target analyte is not amenable to direct conjugation to a carrier protein.     

Peripheral blood serum from healthy donors contains antibodies against the fragment of transcribed region of ribosomal repeat

Enzyme immunoassay showed that blood serum from healthy donors contains specific high-affinity antibodies (apparent association constant ≥5×10⁹ M^–1) against a fragment of transcribed region of ribosomal DNA repeat of human serum, which are present in a free form or are bound to extracellular DNA. Preheating of the serum at 55°C and high ionic strength (1.5 M NaCl) had no effect on the interaction of antibodies with this fragment. Competitive binding assay showed that these antibodies recognize DNA epitopes, which differ from the epitopes recognized by most anti-DNA antibodies in systemic lupus erythematosus. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 277–281, September, 2007     

Rous sarcoma virus p19 binds to specific double-stranded regions of viral RNA: effect of p19 on cleavage of viral RNA by RNase III.

The extent of binding of purified RSV(Pr-C) p19 and p12 to a variety of RNAs was measured using a sensitive nitrocellulose filter binding assay which is capable of detecting binding reactions with association constants as low as 3 × 10⁶ liters × mol^?1 (Hizi, A., Leis, J. P., and Joklik, W. K. 1977). RSV p19 bound 60 and 34 S RSV (Pr-C) RNA with association constants of 5.1 × 10¹¹ and 1.8 × 10¹⁰ liters × mol^?1. RSV p19 bound preferentially to specific double-stranded regions of the RNA since: (a) The association constant for Neurospora nuclease-digested 34 S RNA was the same as for untreated RNA; (b) the association constant for 34 S RNA partially digested with Escherichia coli RNase III (which is specific for double-stranded RNA regions) was 30-fold lower than for untreated RNA; (c) p19 prevented cleavage of 34 S RSV-RNA by E. coli RNase III; (d) p19 bound cell precursor RNAs containing RNase III-sensitive sites, but not mature RNAs lacking RNase III-sensitive sites. On the other hand, purified RSV p12 bound all RNAs tested with association constants roughly proportional to their molecular weights. A possible function for p19 in regulating the processing of viral RNA and its subsequent translation has been proposed.