Molecular cloning of infectious pancreatic necrosis virus and characterization of the coding capacity of the complementary DNA Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/707959971

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  • The two segments of double-stranded RNA (dsRNA) from infectious pancreatic necrosis virus (IPNV) were cloned into plasmid vector pUC8. Two distinct sets of overlapping clones were identified by restriction enzyme and Southern blot analysis. Each of these sets was shown, by Northern blot analysis, to be exclusively related to either segment A or B of genomic RNA. The synthesis of complementary DNA (cDNA) from viral RNA segments A and B was less than full-length. Continuous sequences from each segment were represented by two overlapping inserts which were ligated together at a common restriction site. The entire lengths of the cloned segments A and B were estimated to be 2.9 and 2.6 kilobases, respectively. The cDNA sequences from segements A and B were subcloned into the T7 RNA polymerase vectors, pT71 and pT72 and used to transcribe single-stranded RNA (ssRNA). This RNA was used to indirectly compare the lengths of the cloned sequences to those of the viral dsRNA by glyoxal denaturation and agarose gel electrophoresis. The electrophoretic mobilities of the single-stranded RNA's originating from cloned viral sequences were identical to those of the individual strands of ds genomic RNA. The coding capacity of the viral cDNA was determined by cell-free translation analysis. The single-stranded RNA described above was active in a rabbit reticulocyte lysate translation system and did not require either 5'-capping or 3'-polyadenylation. The electrophoretic mobility of the proteins originating from the cloned viral segments was compared to those produced from viral dsRNA as well as the proteins found in purified virus. The four proteins reportedly encoded by the genome of IPNV were identified among the translation products of the individual cloned segments by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The coding assignments of the proteins produced from cloned segments A and B are shown to be identical to those previously reported for IPNV.
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