The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family.
The amino terminal sequences of five light and heavy immunoglobulin chains from myeloma proteins of the BALB/c mouse with binding activity to phosphorylcholine are presented. Except for a single substitution in position 4, all five heavy chains have identical amino terminal sequences through the first hypervariable region. Proteins which share unique (idiotypic) antigenic determinants are identical through the first hypervariable region of their light and heavy chains. Proteins with differing idiotypic determinants have light chains of differing amino acid sequence. These observations suggest that the heavy chain plays a more important role than the light chain in determining the phosphorylcholine binding site.
Two types of oligonucleotides were synthesized with linker groups attached at the 5'-end. Both were repeating dimers of deoxyribocytidine and deoxyriboadenosine. A 20-mer was prepared with a thiol-containing linker, masked as a disulfide, and a 50-mer was prepared with a vicinal diol-containing linker. A tetraiodoacetylated poly(ethylene glycol) (PEG) derivative was synthesized and reacted with the thiol-containing 20-mer to provide an oligonucleotide PEG conjugate of precisely four oligonucleotides on each PEG carrier. The vicinal diol on the 50-mer was oxidized to an aldehyde and conjugated to keyhole limpet hemocyanin (KLH) to provide an oligonucleotide-KLH conjugate by reductive alkylation. The conjugates were annealed with complementary (TG)n strands. While the double-stranded oligonucleotide-KLH conjugate is an immunogen, eliciting the synthesis of antibodies against oligonucleotides, the PEG conjugate has the biological property of specifically suppressing (tolerizing) B cells which make antibodies against the immunizing oligonucleotide.
We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.
A discrete tetravalent conjugate, 7a (LJP 394), consisting of four oligonucleotides attached to a common carrier or platform was prepared. Single-stranded oligonucleotide 20-mers consisting of alternating deoxycytidine-deoxyadenosine nucleotides, (CA)10, were attached to a tetrabromoacetylated platform by displacement with sulfhydryl-terminated linkers. The tetrabromoacetylated platform 3a was synthesized in three steps using triethylene glycol bis-(chloroformate). The single-stranded conjugate was characterized by polyacrylamide gel electrophoresis, DNA sequencing, phosphate analysis, carbon and nitrogen combustion analysis, and correlation of stoichiometry to conversion in the conjugation process. HPLC and capillary electrophoretic methods were developed to evaluate purity. The tetrakis, single-stranded conjugate was annealed with a stoichiometric amount of a complementary single-stranded oligonucleotide 20-mer consisting of alternating thymidine-deoxyguanosine nucleotides, (TG)10. The double-stranded conjugate LJP 394 was characterized by melt temperature and hyperchromicity, phosphate analysis, and carbon and nitrogen combustion analysis. LJP 394 inhibits binding of DNA to anti-double-stranded oligonucleotide antibodies and reduces anti-oligonucleotide-specific plaque (antibody)-forming cells in an immunized mouse model by a proposed mechanism involving cross-linking B cell surface immunoglobins.
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