Loop size in newt lampbrush chromosomes

Lampbrush chromosome 11 from the newtTaricha granulosa was studied by scanning electron microscopy (SEM) to determine the size of all loops in this bivalent. Measurements with an XY digitizer revealed a mean loop length of 14.9 µm, with a large standard deviation and a skewed distribution toward higher values. The size of the loops at this stage of diplotene extension is similar to that reported in other eukaryotes studies with different approaches. We estimated, from the DNA content of chromosome 11, that between 0.4% and 2.2% of the DNA is found in the loops while the rest of the DNA must remain in the compact chromomeres.     

Monoclonal antibodies specific for thiophosphorylated proteins recognize Xenopus MPF

Maturation promoting factor, (MPF), is a crucial regulatory component of the eukaryotic cell cycle. Though it is ubiquitous, MPF has been difficult to purify to homogeneity, and little is known about its physical properties or composition. In an attempt to further characterize and purify this protein, we have isolated five monoclonal antibodies that immunoadsorb MPF activity, and inhibit the activity in solution. However, all the antibodies recognize many proteins in partially purified MPF. We have shown that antibody binding is dependent on previous exposure of the preparation to ATP gamma S. This suggests that the antibodies specifically recognize thiophosphoproteins, although not all thiophosphorylated proteins in MPF are immunoprecipitated. Using one antibody, MPF was partially purified by immunoadsorption chromatography. These experiments provide the first evidence that MPF from Xenopus is a phosphoprotein that becomes thiophosphorylated upon addition of ATP gamma S.     

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.     

Monoclonal anti-idiotypic antibodies that recognize the binding site for nicotine on rat brain receptor

Anti-idiotypic monoclonal antibodies have been prepared that represent the internal image of nicotine and are specific for the nicotine binding site on rat brain receptor. Specificity of these antibodies for the combining site on anti-nicotine was demonstrated by their ability to inhibit binding of monoclonal anti-nicotine to immobilized nicotine-polylysine. Furthermore, purified rat brain nicotine receptor but not acetylcholine receptor from fish electric organ effectively competed with anti-nicotine for immobilized nicotine and for immobilized anti-idiotype. Only 9 pmoles of naturally occurring (-)-nicotine inhibited idiotype-anti-idiotype binding by 50% whereas 11 times more (+)-nicotine was required. Acetylcholine, several cholinergic agonists and antagonists, nicotine metabolites, and other structurally related compounds were poor inhibitors.     

Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes

Immunization of BALB/c mice with a cadmium-chelate-protein conjugate resulted in the isolation of two hybridoma cell lines (A4 and E5) that synthesized antibodies with different variable regions, but similar metal-chelate affinity. The ability of these two monoclonal antibodies to interact with 12 different metal-chelate complexes was studied using the KinExA 3000 immunoassay instrument. The two antibodies showed the highest affinity for cadmium and mercury complexes of ethylenediamine N,N,N',N'-tetraacetic acid (EDTA). The E5 antibody bound to EDTA complexes of cadmium and mercury with equilibrium dissociation constants (K(d)) of 1.62 x 10(-)(9) M and 3.64 x 10(-)(9) M, respectively. The corresponding values for the A4 antibody were 14.7 x 10(-)(9) M and 3.56 x 10(-)(9) M. Addition of a cyclohexyl ring to the EDTA backbone increased the affinity of E5 for the metal-chelate haptens, while decreasing the binding of A4 to the same haptens. Based on available crystal structures, molecular models were constructed for five different divalent metal-chelate complexes. The models were compared to determine structural features of the haptens that may influence antibody recognition. Difference distance matrixes were used to identify areas of the metal-chelate haptens that differed in three-dimensional space. Antibody affinity correlated well with the extent of total structural difference for these metal-EDTA complexes.     

Characterization of the lampbrush chromosomes of the marbled newt Triturus marmoratus (Latreille, 1800)

The maps of the lampbrush chromosomes of Triturus marmoratus oocytes were constructed on the basis of their lengths and major morphological characters such as giant fusing loops, dense matrix loops, lumpy objects, axial granules, lateral globules and reflected fusions; a nucleolus organizing region occurs subterminally on the right side of chromosome X. — Bivalent I appears morphologically asymmetrical, its two partners being of different lengths and bearing heteromorphic loops and other heterozygous structures: this heteromorphism may indicate that the two partners of bivalent I represent the ZW heterochromosomes of the species. Finally, an autoradiographic study has been performed in order to ascertain the pattern of ³H-uridine incorporation shown by the most typical landmarks and nucleoli.This work was financially supported by C.N.R., Roma.     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.