Gene expression, genome organization, and evolution of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vx021h73s

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  • The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was mapped by examining overlapping Hindlll fragments from cosmid clones which had been constructed from partial Hindlll digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hybridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica (AcMNPV). The hybridization pattern indicated that the genomes of these viruses are similarly organized. In order to examine a homologous region in greater detail, a ³²P-labeled cloned DNA fragment (AcMNPV Hindlll-Q) containing one of the repeated sequences and a portion of the p10 gene from AcMNPV was used to probe Southern blots containing restriction endonuclease digests of AcMNPV DNA, and OpMNPV DNA. A single 3.6 Kb fragment, OpMNPV HindIII-Q was hybridized. The OpMNPV HindIII-Q fragment was cloned into pUC-18, mapped with restriction endonucleases, and reprobed with the AcMNPV Hindlll-Q fragment. A small region of ca. 700 base-pairs, near the left end of the cloned fragment, was cross-hybridized. DNA sequencing in this region revealed an open reading frame of 279 base-pairs which had a detectable homology to the p10 gene of AcMNPV. The sequences upstream and downstream from the p10 gene in both viruses contain long open reading frames which also share a high degree of homology. Northern blot analysis of RNA from OpMNPV infected Orgyia leucostigma cells was used to define the temporal and spatial organization of transcripts from this region. Overlapping mRNAs were found. S1 analysis of the termini of the major p10 mRNA indicates nontranslated regions of 52-53 bases at the 5'-end and 176-181 bases at the 3'-end. The 5'-mRNA start site was located within a 12 nucleotide sequence which appears conserved in all late hyper-expressed baculovirus genes. S1 analysis was performed on an early message from this region and the 5'-mRNA start site lies 29 base-pairs downstream from the beginning of a putative TATA box. No homology was detected between OpMNPV DNA and the repeated sequence element present on the AcMNPV HindIII-Q fragment.
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