Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.     

Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen

Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum.     

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.     

Antibody-nucleic acid complexes. Antigenic domains within nucleosides as defined by solid-phase immunoassay

The usefulness of solid-phase immunoassays for the characterization of anti-nucleoside antibodies was investigated. Antibodies specific for guanosine (G), 7-methyl-guanosine (m7G), and cytidine (C) were obtained from the serum of rabbits immunized with nucleoside-KLH (keyhole limpet hemocyanin) conjugates. Solid-phase assays consisted of measuring the ability of these antibodies to be retained by microtiter wells containing immobilized nucleoside-BSA (bovine serum albumin) conjugates. Nucleosides employed as haptens included adenosine (A), N6-methyl-A (m6A), guanosine (G), N2,N2-dimethyl-G (m22G), 1-methyl-G (m1G), O6-methyl-G (m6G), 7-methyl-G (m7G), cytidine (C), 5-methyl-C (m5C), uridine (U), and ribothymidine (T). Spectral analysis of these conjugates revealed that 15-20 nucleosides were coupled to each BSA molecule. Quantitative information regarding the various reactions associated with these assays was obtained by employing antigen and antibody (IgG) preparations radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde (specific activities 0.6-2.1 X 10(6) cpm/micrograms). Data obtained with 3H-labeled antigens indicated that the adsorption of all nucleoside-BSA conjugates was uniform and irreversible with respect to the assay conditions used. Assays designed to measure antibody binding in the presence of excess antigen revealed that (i) nonspecific binding to immobilized BSA was negligible, (ii) as little as 0.5 ng of bound antibody could be detected, (iii) antibody retention was directly proportional to antibody concentration, and (iv) each anti-nucleoside antibody cross-reacted to a considerable extent with nonhomologous haptens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of immunoadsorbent efficiency and capacity for the isolation of tRNAs containing N6-(delta 2-isopentenyl)adenosine derivatives

Antibodies directed to modified nucleosides recognize the nucleoside (antigen) when it is present in an intact tRNA molecule. The general application of anti-nucleoside immunoadsorbent chromatography, however, has been greatly impeded by the apparent inefficiency and low capacity of conventional immunoadsorbents for transfer RNA. Antibodies specific for isopentenyladenosine (i6A) were employed to investigate the efficacy of various immunoadsorbents with respect to immobilization of antibody protein and with respect to their ability to bind i6A-containing tRNAs. Biologically active anti-i6A was recovered in high yield (80-88%) by affinity chromatography on i6A-adipate-Sepharose 4B or i6A-butane diglycidyl ether-Sepharose 4B using either 15% pyridine in phosphate-buffered saline or 0.2 M acetic acid as eluents. The binding capacity of various anti-i6A antibody immunoadsorbents was evaluated. While both anti-i6A antibody-protein A-agarose-iminothiolane (ITL) and anti-i6A antibody-protein A-agarose-dimethyl suberimidate showed a high capacity for i6A-tRNA, the latter column is much less efficient with respect to antibody immobilization. Under optimal conditions, the ITL immunoadsorbent binds 5-6 nmol of i6A/mg of antibody protein. With respect to bulk tRNA, 1 mg of antibody protein (ITL immunoadsorbent) binds all of the i6A-tRNA in a 1-mg sample.     

Production of antisera specific for idiotype(s) of murine anti-(T,G)-A--L antibodies.

Antibodies to the synthetic polypeptide (T,G)-A--L were raised in C57BL/10 and C3H.SW mice. For each strain, the anti-(T,G)-A--L antibodies from 10 animals were pooled, affinity purified on a (T,G)-A--L-Sepharose column, and used to immunize Lewis rats. The resulting rat antisera were adsorbed with insolubilized normal mouse globulin in order to remove anti-isotypic and anti-allotypic antibodies. The residual antibodies specifically inhibited the binding of (T,G)-A--L by anti-(T,G)-A--L as measured by a radioimmunoassay. The specificity of this inhibition was demonstrated as follows: 1) failure of the anti-(T,G)-A--L anti-idiotype to inhibit the binding of nuclease to anti-nuclease antibody of the same allotype; 2) failure of Lewis anti-[B10 anti-(T,G)-A--L] to inhibit C3H.SW anti-(T,G)-A--L and vice versa; 3) ability to absorb anti-C3H.SW anti-idiotypic activity on insolubilized C3H.SW anti-(T,G)-A--L but not on B10 anti-(T,G)-A--L. The same or cross-reactive idiotype(s) was present in the majority of individuals of each of these strains.     

Isolation and characterization of rabbit anti-m7G-5'-P antibodies of high apparent affinity.

Antibodies highly specific for intact pm7G (7methylguanosine-5'-mono-phosphate) were induced by immunization of rabbits with a pm7G-BSA conjugate. Since the nucleotide is six-fold more stable than m7G (7-methylguanosine) to alkali-catalyzed fission of the imidazole ring, it is a more desirable antigen for obtaining antibodies capable of binding caps on eukaryotic mRNA. UV spectra demonstrated that the nucleotide in the conjugate was predominantly the intact form. Competition radioimmunoassay showed 1) high apparent affinities for pm7G, on the order of 10(-8)M, 2) low competition by the ring-opened form of the homologous hapten (*pm7G), and by m7G, 3) little or no competition by AMP, GMP, CMP, UMP or m6A, and 4) high apparent affinities for m7GpppAm, m7GpppN6MAm, m7GpppGm, m7GpppA.     

Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 microM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 microM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Isolation and characterization of rabbit anti-m3 2,2,7G antibodies.

Antibodies specific for intact 2,2,7-trimethylguanosine (m3 2,2,7G) were induced by immunization of rabbits with a nucleoside-human serum albumen (HSA) conjugate. Competition radioimmunoassay showed that the antibody distinguishes well between intact m3 2,2,7G and its alkali-hydrolysed form (m3 2,2,7G*). Antibody specificity is largely dependent on the presence of all three methyl groups in m3 2,2,7G: none of the less extensively methylated nucleosides m7G, m2G and m2 2,2G is able to compete efficiently with the homologous hapten. Little or no competition was observed with m1G, m1A, m6A, m5U and each of the four unmodified ribonucleosides. Binding studies with nucleoplasmic RNAs from Ehrlich ascites cells suggest that the antibody reacts specifically with the m3 2,2,7G-containing cap structure of the small nuclear U-RNAs (U-snRNAs). Thus the antibody should be a valuable tool for studying the role of the 5'-terminal regions of the U-snRNAs of eucaryotic cells.