Biology of a constitutively-expressed vaccinia virus gene required for DNA replication Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/mp48sg41m

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  • Replication of vaccinia virus in the cytoplasm of the infected cell occurs under the direction of virally encoded gene products. Expression of approximately 200 viral genes follows a highly regulated temporal scheme which can be followed biochemically and morphologically during the assembly process. In order to dissect this complex genetic program conditionally lethal mutants of vaccinia have previously been generated. This report describes the utilization of a collection of temperature sensitive (ts) mutants for the study of a required vaccinia virus gene. Previous biochemical analysis defined four biochemical phenotypes within this collection of is mutants: DNA negative, defective-late, abortive-late, and wild-type. One mutant from each phenotype was examined at the non-permissive temperature by electron microscopy. Each exhibited distinct morphological aberrations, but it was difficult to relate aberrant morphology to the biochemical phenotype. Therefore, one mutant, ts17, was chosen for more detailed study. Ts17 is a member of the DNA negative biochemical class. At the non-permissive temperature no viral DNA was produced in ts17 infected cells. Early viral proteins were synthesized for up to 24 hours post-infection, and late viral proteins were not expressed. The ts17 gene was then mapped by marker rescue techniques, and the nucleotide sequence of 3.6 kilobases of DNA form that region was determined. The nucleotide sequence of a fragment from tsi7 viral DNA, and from two ts17 revertants was also determined, and the nature of the tsi7 mutation was identified. Analysis of the wild-type sequence revealed three tightly spaced tandemly-oriented open reading frames. The predicted proteins encoded for by these open reading frames were confirmed by hybrid-selection in vitro translation of selected mRNAs. S1 mapping of the 5' and 3' ends of the encoded transcripts, in conjunction with a northern analysis, determined that two of the open reading frames terminate coincidentally. S1 analysis utilizing RNAs isolated over time demonstrated that the ts17 gene was transcribed throughout infection and is therefore a constitutive viral gene. Precise mapping showed the transcriptional start site of this gene to be in a proposed late regulatory element.
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