Large RNase T1-resistant oligonucleotides encoding p15E and the U3 region of the long terminal repeat distinguish two biological classes of mink cell focus-forming type C viruses of inbred mice

We used T1 oligonucleotide maps, in conjunction with available nucleotide sequences of appropriate C-type viruses, to identify regions of the viral genome that distinguish two biological classes of mink cell focus-forming (MCF) viruses described previously by Cloyd et al. (J. Exp. Med. 151:542-522, 1980). We found that leukemogenic MCF viruses from thymus differed from non-leukemogenic MCFs isolated from nonthymic neoplasms in nucleotide sequences encoding Prp15E and the U3 portion of the long terminal repeat (LTR). The thymic isolates possessed recombinant Prp15E genes, with the 5' to mid portion derived from their ecotropic parents and the extreme 3' portion invariably derived from their nonecotropic parents. These viruses probably derived the entire U3 portion of their LTRs from their nonecotropic parents. The nonthymic MCFs appeared to inherit their entire Prp15E coding region from their nonecotropic parents. We failed to detect consistent differences in gp70-coding sequences between the two groups of MCFs, but this may simply reflect limitations of the data. The studies presented here, in conjunction with studies from a number of labs indicating a role for MCF gp70 in leukemogenesis, indicate that three genetic elements, gp70, p15E, and the U3 portion of the LTR, may all play a role in determining the leukemogenic phenotype of type C viruses of high-leukemic inbred mice.     

Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10³ and 10⁴ times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D⁵) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K⁵). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.     

Genbank accession #: AF 124983

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