- Volume 68, Issue 1, 1987
Volume 68, Issue 1, 1987
- Animal
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Monoclonal Antibodies to Cowpox Virus: Polypeptide Analysis of Several Major Antigens
SUMMARYMonoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.
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Stability of a Bacterial Gene in a Bovine Papillomavirus-based Shuttle Vector Maintained Extrachromosomally in Mammalian Cells
More LessSUMMARY
In order to analyse the stability of cloned genes in a viral vector we have constructed a shuttle vector based on bovine papillomavirus and the Escherichia coli gene lacZ. Propagation of this vector in mouse C127 cells and analysis of vector sequences in bacteria produced no detectable mutations in the lacZ gene in over 6137 clones analysed. This is 100-fold less than the mutation frequency observed when the same and similar target genes are replicated in monkey COS cells using a simian virus 40- based shuttle vector.
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Characterization of the Genome of Soybean Chlorotic Mottle Virus
More LessSUMMARYThe DNA of soybean chlorotic mottle virus (SoyCMV) has been isolated. When analysed by gel electrophoresis, the native DNA formed multiple bands resembling DNA from other caulimoviruses. The viral DNA was cloned in bacterial vectors. Cloned DNA was used to map restriction sites on the SoyCMV DNA. Comparison of cloned DNAs of SoyCMV and cauliflower mosaic virus (CaMV) by Southern transfer hybridization revealed the absence of significant sequence homology. Electrophoresis in denaturing gels showed that SoyCMV DNA had three single-strand discontinuities (‘gaps’), one in one strand and two in the other, and the positions of these gaps were mapped on the viral DNA. Potential primer sites for reverse transcription were detected in the cloned DNA by sequence analysis of regions around the gap sites. The results obtained definitely establish SoyCMV as a member of the caulimovirus group and suggest a replication mechanism for this virus similar to that described for CaMV.
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Cell-free Translation of Turnip Mosaic Virus RNA
More LessSummaryRNA from the type strain of turnip mosaic virus (TuMV), a member of the poty virus group, was translated in vitro in either rabbit reticulocyte lysate or wheat germ extract. We found no evidence for encapsidated, subgenomic TuMV RNA species. A broad size range of l-[3SS]methionine-labelled polypeptides with molecular weights up to 200000 (200K) was observed in TuMV RNA-programmed reticulocyte lysates. In contrast, a single polypeptide of 44K predominated in the much narrower spectrum of labelled products, of 55K or less, seen in wheat germ extracts containing TuMV RNA. Time course experiments revealed that, in rabbit reticulocyte incubations, many of the low molecular weight TuMV RNA-encoded polypeptides accumulated only after synthesis of the largest polypeptides (i.e. those ⩾100K). When the amino acid analogues p-fluorophenylalanine, l-canavanine and N-tosyl-lysyl-chloromethane were substituted for phenylalanine, arginine and lysine respectively, the major 44K product detected in wheat germ extracts diminished, to be replaced by a product of 92K which co-migrated with the major polypeptide normally observed in reticulocyte incubations. A rapid processing event may occur in standard wheat germ incubations to release the 44K polypeptide from a nascent, elongating precursor. The 44K product did not arise because of limiting amounts of a particular tRNA species. Reticulocyte incubations with or without additional reducing agent showed comparable levels of all the processed TuMV RNA-specific products. We could detect no serological crossreaction between any of the TuMV RNA-encoded products and antiserum raised against HPLC-purified helper component of tobacco vein mottling virus, another potyvirus.
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Characterization of the Coat Protein Subgenomic RNA of White Clover Mosaic Virus
More LessSummaryThe genomic RNA (6·2 kb) and a 0·9 kb RNA were detected by Northern hybridization in leaves infected with white clover mosaic virus (WC1MV). The 0·9 kb RNA was efficiently encapsidated by one, but not by eight other, isolates of WC1MV. Both RNA species were shown to possess 3′-terminal tracts of poly (A) 200 to 300 nucleotides long by end-labelling with [32P]pCp and digestion with ribonucleases A, Phy M, T, and U2. The 0·9 kb RNA was mapped to the 3′-terminal region of the genomic RNA using cDNA probes. In rabbit reticulocyte lysates, the 6·2 kb RNA directed predominantly the synthesis of a protein of mol. wt. 160000 (160K) and several additional proteins. The 0·9 kb RN A directed synthesis of a 25K protein. This protein was shown to be the virus coat protein by its co-migration in gels with the WC1MV coat protein and by its specific immunoprecipitation with antiserum to virus particles. Both RNA species were found in specific association with polysomes indicating that they function as messenger RNAs in infected cells.
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Unilateral Compatibility of Genome Segments from Two Distinct Strains of Red Clover Necrotic Mosaic Virus
More LessSUMMARYTwo strains of red clover necrotic mosaic virus (TpM 34 and TpM 48) were distinguished by their serological relationship, by the symptoms they induced in selected host plants and by Northern hybridization analysis. Purified RNA 1 and RNA 2 of the two strains were inoculated to the leaves of Chenopodium amaranticolor, Vigna unguiculata and C. quinoa in all possible combinations. It was demonstrated that a heterologous mixture containing RNA 1 of TpM 34 and RNA 2 of TpM 48 was infectious, resulting in lesion development whereas the reciprocal combination was not. The infectious pseudorecombinant was isolated by several single lesion transfers in C. quinoa and its genetic nature was confirmed by serology and Northern hybridization analysis. Inoculation of the pseudorecombinant and the two parental strains to five selected host plants revealed that symptom expression was determined by RNA 1.
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