Genomic comparisons of seed-transmissible and non-seed-transmissible strains of cucumber mosaic virus Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/sx61dq83k

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  • A sensitive assay for the analysis of contaminating segments in pseudorecombinants constructed from a seed-transmissible (CVM-Pg) and a non-seed-transmissible (CMV-Le) strain of cucumber mosaic virus was developed using partial RNase T₁ digestion. Isolated genomic RNA segments produced unique digestion patterns when 3'- end labeled, treated with RNase T₁, and subjected to polyacrylamide gel electrophoresis. Although unique bands were present in these patterns, an overall similarity existed, resulting from putative insertions and deletions in the RNA of each strain. Northern blot hybridization studies of the homology between CMV-Pg and CMV-Le indicated that all 4 heterologous RNA segments contained sequences that were greater than 83% homologous. Homology between the total RNA from each strain was estimated at 70% when assayed by S₁ nuclease digestion following hybridization of RNA to a complementary DNA probe under stringent conditions. A comparison of the electrophoretic mobility of each RNA of both strains in agarose, glyoxal and polyacrylamide gels suggested that each segment was identical in size. RNA segments 1 and 2 when compared to the heterologous strain, however, had discernable differences in the secondary structure. A 5th RNA was detected in both strains. It migrated as two distinct bands when subjected to polyacrylamide gel electrophoresis, and was 3'-end labeled preferentially when present with segments 1-4. Complementary DNA synthesized from RNA 5 of one strain hybridized to segments 1-4 of both strains as determined by northern blot hybridization. RNA 5 of both strains produced identical patterns when subjected to partial RNase T₁ digestion, yet they differed significantly when compared to the digestion patterns from RNA 4 of the homologous strain. These results indicated that RNA 5 of both strains contained virtually identical nucleotide sequences and that they originated from RNA's 1-4, but not from the extreme 3'-terminus. Total CMV RNA, subjected to RNase H treatment after hybridization to a specific deoxyribohexanucleotide, produced two novel bands which were visualized by ethidium bromide staining. Double-stranded cDNA synthesized from RNA of each strain, cloned by blunt-end ligation, produced recombinant clones that contained only inserts of insignificant size.
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