ABSTRACT
We analyzed VDJ and VJ rearrangements in IgM-secreting B lymphocytes from a cow infected with bovine leukemia virus (BLV). BLV causes expansion of CD5(+) and IgM(+) B lymphocytes regardless of antigen specificity. The data showed that single point mutations contribute to the diversification of IgM antibodies. The most striking observation, however, is that approximately 9% of theVDJ rearrangement in IgM-secreting B cells encode an exceptionally long third complementarity-determining region of the heavy chain (CDR3H; 56 to 61 amino acids) with multiple cysteine residues. Such an exceptionally long CDR3H is the first ever to be documented for an antibody in a species. These VDJ rearrangements encode functional IgM antibodies as some of these show polyspecific reactivity. The presence of even-numbered cysteine residues in the CDR3H may provide hitherto unknown configurational ability to the antigen combining site via intra-CDR3H disulfide bridging. In addition, the VDJ rearrangements encoding exceptionally long CDR3H paired with either novel V(lambda)1 or V(x)1x genes, earlier noted not to be expressed. Overall, these experiments provide evidence that somatic hypermutations and generation of an exceptionally long CDR3H contribute to the diversification of IgM antibodies in cattle.
Subject(s)
Cattle/genetics , Cattle/immunology , Genes, Immunoglobulin , Immunoglobulin M/genetics , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Antibody Diversity , Base Sequence , Cysteine/chemistry , DNA Primers/genetics , Enzootic Bovine Leukosis/genetics , Enzootic Bovine Leukosis/immunology , Gene Rearrangement, B-Lymphocyte , Genes, env , Hybridomas , Immunoglobulin M/chemistry , Immunoglobulin Variable Region/chemistry , Leukemia Virus, Bovine/genetics , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5-tert-octyl, 5-cumyl, 5-(4-tert-butylphenyl, 5-(1-adamantyl), 5-(n-dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420-435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8-14 carbon substitutents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera. and a 455-nm cutoff optical filter.
Subject(s)
DNA/analysis , Nucleic Acid Hybridization , Oligonucleotide Probes , Phosphates/chemical synthesis , Salicylates/chemistry , Alkaline Phosphatase/metabolism , Animals , Base Sequence , Fluorescence , Hydrolysis , Intestines/enzymology , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Plasmids , Substrate SpecificityABSTRACT
Synthetic procedures are presented for a new chelator that forms stable and highly fluorescent complexes with Eu3+. This chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) is synthesized in a high-yield three-step procedure. BCPDA can be covalently incorporated into proteins under relatively mild conditions, and when complexed with Eu3+ forms a fluorescent product that has a lifetime in the range of 0.4 to 0.7 ms. Thus, it is useful for time-resolved fluorescence immunoassay applications.
